JP5843840B2 - New cancer marker - Google Patents

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JP5843840B2
JP5843840B2 JP2013236001A JP2013236001A JP5843840B2 JP 5843840 B2 JP5843840 B2 JP 5843840B2 JP 2013236001 A JP2013236001 A JP 2013236001A JP 2013236001 A JP2013236001 A JP 2013236001A JP 5843840 B2 JP5843840 B2 JP 5843840B2
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methylation
cpg sites
cancer
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JP2014036672A (en
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イー. スコテイム,ロルフ
イー. スコテイム,ロルフ
アー. ロテ,ラグンヒルド
アー. ロテ,ラグンヒルド
エー. リンド,グロ
エー. リンド,グロ
セー. アールクイスト,テリェ
セー. アールクイスト,テリェ
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オスロ ユニベルシテートシケヒュース ホーエフ
オスロ ユニベルシテートシケヒュース ホーエフ
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/118Prognosis of disease development
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/154Methylation markers

Description

  The present invention relates to a novel marker for hypermethylation of gene promoters in cancer. In particular, the present invention relates to a method for determining whether a tumor has developed in the airway-digestive system or whether a subject has relapsed after treatment of such a tumor. The method of the invention comprises measuring the methylation status of CpG sites within one or more promoter regions / sequences of a particular gene. The invention further relates to the use of such methylated genes and to diagnostic kits for detecting cancer.

  Epigenetic gene dysregulation is as common as gene mutations in human cancer. These mechanisms lead to quantitative and qualitative gene expression changes that are responsible for the selective growth advantage that can lead to cancerous transformation. An abnormally hypermethylated CpG island within a gene promoter that is associated with transcriptional inactivation is one of the most common epigenetic changes in cancer. Since early detection of disease can lead to improved clinical outcomes for most types of cancer, the identification of abnormal gene methylation associated with cancer represents a promising new biomarker. For cancers in the airway-digestive system, including colorectal cancer, early studies have identified the presence of abnormally methylated DNA in the patient's blood and feces. Genes that are abnormally hypermethylated frequently in benign tumors, and very rarely in normal mucosa, have potential clinical potential for early detection of high-risk adenomas and low-risk stage cancers It would be a good candidate for a diagnostic biomarker because of its profit.

  However, in general, the sensitivity and specificity of existing early markers for airway-digestive cancers are still poor, so only some of the genes that have been screened for methylation so far have been quite significant. It showed high sensitivity and specificity. Specific hypermethylation was seen in VIM (vimentin) and SFRP2 reported by Muller et al. And Chen et al., And recently in a report from Lind et al., While the frequency of hypermethylation of NR3C1 was significantly lower. These results suggest that ADAMTS1 and CRABP1 have a high frequency of cancer-specific hypermethylation in colorectal tumors. Lind et al. Further identified 18 genes as potential markers for colorectal cancer. In a study by Mori et al., The T cell differentiation protein MAL, one of 18 candidate genes discussed by Lind et al., Was methylated with a MAL methylation frequency of 6% (2/34 samples). It was concluded that it would not be a suitable diagnostic biomarker for airway-gastrointestinal cancer. Further analysis of some of the 18 candidate genes performed by the inventor did not give promising results: NDRG1 was not methylated in all samples analyzed and NR3C1 was methylated However, only at a very low frequency, and subsequent sequence analysis of SDHA is unable to confirm the identity of this gene, leaving it unsuitable as a marker for cancer development. It was.

In conclusion, there was no evidence that any of the genes discussed by Lind et al would provide any improvement over current technology for cancer detection. As a result, there is a need for a gene panel in which each gene is hypermethylated with high frequency and specificity in cancer. In particular, there is a need for gene panels that are useful in non-invasive techniques such as techniques involving the use of stool samples, or techniques that can be used on easily obtained sample material such as blood or mucus. Such a gene panel would greatly improve the potential for early detection of these cancers. The ultimate goal would be to develop diagnostic tests that measure hypermethylation of just a few high frequency genetic markers, such as two or three.
Therefore, it is desirable to identify additional genes in which CpG islands within the promoter region are frequently hypermethylated in cancer.

  The present invention recognizes that the inventor recognizes that a particular subset of genes identified as potential markers by Lind et al. Contain CpG sites that are exceptionally frequently methylated in airway-digestive cancers. Is based.

  Thus, it is an object of the present invention to provide a panel of diagnostic markers for cancer, eg, cancer in the respiratory tract-digestive system, particularly colon cancer and colorectal cancer. This panel of markers solves the problems associated with the low specificity and frequency of methylation in the majority of known cancer markers.

Accordingly, one aspect of the present invention is a method for determining whether a subject has developed, is developing, or is likely to develop cancer, or whether the subject has recurred after cancer treatment, comprising: Step:
a) Methylation level of CpG sites, number of CpG sites methylated, or methylation status in the nucleic acid sequence of the promoter region, first exon or intron of at least one gene in a sample obtained from the above-mentioned subject Measuring, wherein the gene is:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
FBN1, for example, identified by ensembl gene ID ENSG000000161477, entrez ID 2200;
SNCA, for example as specified by ensemble gene ID ENSG0000013335, entrez ID 6622; and INA, for example, selected from the group consisting of those specified by ensembl gene ID ENSG00000148798, entrez ID 9118.

The method comprises the following steps:
b) compare the methylation level, the number of methylated CpG sites, or the methylation status of the CpG site to the reference; and c) the methylation level, the number of methylated CpG sites, or CpG If the methylation status of the site is higher than the above standard methylation level, the number of methylated CpG sites, or the methylation status of the CpG site, the subject is more likely to develop or develop cancer. Or is likely to develop, or has recurred after cancer treatment, and the methylation level, number of methylated CpG sites, or methylation status of CpG sites is If the subject's methylation level, number of CpG sites methylated, or methylation status of the CpG site is below the standard, the subject is less likely to develop cancer, has not developed cancer, or develops It can further include determining that the likelihood is low or has not recurred after cancer treatment.

Another aspect of the present invention is a method for determining whether a subject has developed, has developed or is likely to develop cancer, or whether the subject has relapsed after cancer treatment, and includes the following steps: :
a) measuring the methylation level of the sample from the subject, the number of methylated CpG sites, or the methylation status of the CpG sites;
b) creating a percentile plot of the methylation level of the at least one gene obtained from a sample from a healthy population, the number of CpG sites methylated, or the methylation status of CpG sites;
c) Methylation level measured in healthy population, number of methylated CpG sites, or methylation status of CpG sites, and methylation level measured in populations suffering from cancer, methylated CpG sites A ROC (Receiver Operating Characteristic) curve based on the number of or CpG site methylation status;
d) selecting the desired combination of sensitivity and specificity from the ROC curve;
e) determining from the percentile plot the methylation level corresponding to the determined or selected specificity, the number of methylated CpG sites, or the methylation status of the CpG sites; and f) at least one of the above mentioned in the sample The methylation level of one gene, the number of methylated CpG sites, or the methylation status of the CpG sites corresponds to the desired combination of sensitivity / specificity described above, the number of methylated CpG sites Or if it is equal to or higher than the methylation status of the CpG site, it is predicted that the subject is likely to suffer from cancer, and the methylation level of the sample, methylated CpG The number of sites, or the methylation status of the CpG site, represents the methylation level, methylated Cp corresponding to the desired sensitivity / specificity combination described above. If the number of G sites or the methylation status of the CpG sites is low, the subject is less likely to have cancer or is predicted not to have cancer.

The present invention is a diagnostic kit for determining cancer, comprising:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG 20, for example, those identified by ensemble gene ID ENSG0000013104, entrezid 23111;
FBN1, for example, identified by ensembl gene ID ENSG000000161477, entrez ID 2200;
SNCA, eg complementary to the nucleic acid sequence of a gene selected from ensemble gene IDs ENSG0000013335, entrez ID 6622; and INA, eg selected from ensembl gene IDs ENSG000000014798, entrez ID 9118 Further related to the kit comprising one or more oligonucleotide primers or a set of one or more oligonucleotide primers.

The invention also relates to a method of using the marker according to the invention. Thus, the present invention provides: the level of methylation, the number of methylated CpG sites, or the methylation status of CpG sites, whether the subject has developed, is or is likely to develop cancer, or In a diagnostic assay that evaluates as an indicator of whether a subject has relapsed after cancer treatment:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
FBN1, for example, identified by ensembl gene ID ENSG000000161477, entrez ID 2200;
One or more genes selected from the group comprising SNCA, for example those identified by ensembl gene ID ENSG00001333535, entrez ID 6622; Further related to the usage of.

In addition, the present invention provides the level of methylation, the number of methylated CpG sites, or the methylation status of CpG sites, whether the subject has developed, is or is likely to develop cancer, or A method of using a nucleic acid sequence in a diagnostic assay to evaluate as an indicator of whether a subject has relapsed after cancer treatment, wherein the nucleic acid described above is:
A) a nucleic acid sequence defined by any of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 16;
B) a nucleic acid sequence that is complementary to the sequence defined in A);
C) a partial sequence of the nucleic acid sequence defined in A) or B);
D) relates to said method comprising a nucleic acid sequence selected from the group consisting of nucleic acid sequences which are at least 75% identical to the sequences defined in A), B) or C).

The present invention includes the following:
A) a nucleic acid sequence defined by any of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 16;
B) a nucleic acid sequence that is complementary to the sequence defined in A);
C) a partial sequence of the nucleic acid sequence defined in A) or B);
D) Further provided is an antibody that recognizes a methylated nucleic acid sequence selected from the group consisting of nucleic acid sequences that are at least 75% identical to the sequences defined in A), B), or C).

Shown are representative methylation specific polymerase chain reactions resulting from analysis of MAL in three normal mucosal samples, three adenomas, and three carcinomas. The visible PCR product in lane U indicates the presence of an unmethylated allele, whereas the PCR product in lane M indicates the presence of a methylated allele. Abbreviations: A, adenoma; C, carcinoma; N, normal mucosa; POS, normal blood (unmethylated sample control) and in vitro methylated DNA (methylated sample control) positive control; NEG, (template Negative control; U, lane for unmethylated MSP product; M, lane for methylated MSP product. Since the adenomas were run on separate gels, the illustration is a compilation of two gel panels. Figure 5 shows upregulation of gene expression after epigenetic drug treatment of the first methylated cell line. Upregulation of mRNA expression of CNRIP1, INA, and SPG20 is detected in colon cancer cell lines after treatment with demethylated 5-aza-2'-deoxycytidine alone and in combination with the deacetylase inhibitor trichostatin A It was seen. The panel shows 5-aza-2′-deoxycytidine (1 uM and 10 uM) alone, trichostatin A alone, and a combination of the two drugs (1 μM 5-aza-2′-deoxycytidine alone and 0.5 μM trichostatin. The relative expression values of CNRIP1, INA, and SPG20 (equal scale), respectively, in the six colorectal cancer cell lines, HT29, SW48, HCT15, SW480, RKO, and LS1034 treated with A) are demonstrated. Two doses of 5-aza-2'-deoxycytidine (low and high) result in a comparable increase in the relative expression values of all three genes. This means that the demethylation of the cell line is achieved by culturing them in the presence of a low dose of 5-aza-2'-deoxycytidine, the low dose being the cell of this drug It is advantageous in view of toxicity. For CNRIP1 and INA, the combined treatment was more effective than individual treatment with 5-aza-2'-deoxycytidine alone and trichostatin A alone. Combination treatment also enhanced SPG20 expression, however, similar or higher reactivation could be achieved with 5-aza-2'-deoxycytidine treatment alone. As expected, treatment with the deacetylase inhibitor trichostatin A alone did not enhance either CNRIP1, INA, or SPG20 gene expression. Abbreviations: AZA, 5-aza-2'-deoxycytidine; TSA, trichostatin A. Figure 5 shows upregulation of gene expression after epigenetic drug treatment of the first methylated cell line. Upregulation of mRNA expression of CNRIP1, INA, and SPG20 is detected in colon cancer cell lines after treatment with demethylated 5-aza-2'-deoxycytidine alone and in combination with the deacetylase inhibitor trichostatin A It was seen. The panel shows 5-aza-2′-deoxycytidine (1 uM and 10 uM) alone, trichostatin A alone, and a combination of the two drugs (1 μM 5-aza-2′-deoxycytidine alone and 0.5 μM trichostatin. The relative expression values of CNRIP1, INA, and SPG20 (equal scale), respectively, in the six colorectal cancer cell lines, HT29, SW48, HCT15, SW480, RKO, and LS1034 treated with A) are demonstrated. Two doses of 5-aza-2'-deoxycytidine (low and high) result in a comparable increase in the relative expression values of all three genes. This means that the demethylation of the cell line is achieved by culturing them in the presence of a low dose of 5-aza-2'-deoxycytidine, the low dose being the cell of this drug It is advantageous in view of toxicity. For CNRIP1 and INA, the combined treatment was more effective than individual treatment with 5-aza-2'-deoxycytidine alone and trichostatin A alone. Combination treatment also enhanced SPG20 expression, however, similar or higher reactivation could be achieved with 5-aza-2'-deoxycytidine treatment alone. As expected, treatment of the deacetylase inhibitor trichostatin A alone did not enhance either CNRIP1, INA, or SPG20 gene expression. Abbreviations: AZA, 5-aza-2'-deoxycytidine; TSA, trichostatin A. Figure 5 shows upregulation of gene expression after epigenetic drug treatment of the first methylated cell line. Upregulation of mRNA expression of CNRIP1, INA, and SPG20 is detected in colon cancer cell lines after treatment with demethylated 5-aza-2'-deoxycytidine alone and in combination with the deacetylase inhibitor trichostatin A It was seen. The panel shows 5-aza-2′-deoxycytidine (1 uM and 10 uM) alone, trichostatin A alone, and a combination of the two drugs (1 μM 5-aza-2′-deoxycytidine alone and 0.5 μM trichostatin. The relative expression values of CNRIP1, INA, and SPG20 (equal scale), respectively, in the six colorectal cancer cell lines, HT29, SW48, HCT15, SW480, RKO, and LS1034 treated with A) are demonstrated. Two doses of 5-aza-2'-deoxycytidine (low and high) result in a comparable increase in the relative expression values of all three genes. This means that the demethylation of the cell line is achieved by culturing them in the presence of a low dose of 5-aza-2'-deoxycytidine, the low dose being the cell of this drug It is advantageous in view of toxicity. For CNRIP1 and INA, the combined treatment was more effective than individual treatment with 5-aza-2'-deoxycytidine alone and trichostatin A alone. Combination treatment also enhanced SPG20 expression, however, similar or higher reactivation could be achieved with 5-aza-2'-deoxycytidine treatment alone. As expected, treatment of the deacetylase inhibitor trichostatin A alone did not enhance either CNRIP1, INA, or SPG20 gene expression. Abbreviations: AZA, 5-aza-2'-deoxycytidine; TSA, trichostatin A. The methylation status of the MAL promoter in normal colon mucus samples and colorectal cancer is shown. Representative results from a methylation specific polymerase chain reaction are shown. The visible PCR product in lane U indicates the presence of an unmethylated allele, while the PCR product in lane M indicates the presence of a methylated allele. N, normal mucosa; C, carcinoma; Pos, positive control (unmethylated reaction: DNA from normal blood, methylation reaction: methylated DNA in vitro); Neg, negative control (including water as template); U Unmethylated MSP product lane; M, Methylated MSP product lane. Shows site-specific methylation within the MAL promoter. Bisulfite sequencing of the MAL promoter verifies the methylation status assessed by methylation-specific polymerase chain reaction. The top of the figure is a schematic representation of the CpG sites successfully amplified by the two analytical bisulfite sequencing fragments A (−68 to +168; to the right) and B (−427 to −85; to the left). The transcription start site is represented by +1 and the vertical bars indicate the position of individual CpG sites. Two arrows indicate the position of the MSP primer. With respect to the bottom of the figure, black circles represent methylated CpGs; white circles represent unmethylated CpGs; and hatched white circles represent partially methylated sites (about 20-80% cytosine in addition to thymine). Present). The lower right U, M, and U / M columns list the methylation status of each cell line evaluated by us using MSP analysis. Abbreviations: MSP, methylation specific PCR method; s, sense; as, antisense; U, unmethylated; M, methylated; U / m, presence of both unmethylated and methylated bands. 2 shows “bisulfite sequencing” of the MAL promoter. Electropherogram of representative bisulfite sequencing of the MAL promoter of a colon cancer cell line. Subsection of electropherogram for bisulfite sequencing over a range of CpG sites from +11 to +15 relative to the transcription start. Cytosine within the CpG site is indicated by a black arrow, while cytosine converted to thymine is underlined in red. The MAL promoter sequencing electropherograms illustrated here are from the unmethylated V9P cell line, as well as from hypermethylated ALA and HCT116. MAL expression in cancer cell lines and colorectal cancer. MAL promoter hypermethylation was associated with decreased or lost gene expression in an in vitro model. MAL quantitative gene expression levels are expressed as the ratio between the average of the two MAL assays (detecting various splice variants) and the average of the two endogenous controls, GUSB and ACTB. That value was multiplied by a factor of 1000. At the bottom of each sample, the respective methylation status as assessed by methylation specific polymerase chain reaction is shown. Black circles represent MAL promoter hypermethylation, white circles represent unmethylated MAL, and hatched white circles represent the presence of both unmethylated and methylated alleles. Colorectal cancer is divided into an unmethylated group (n = 3) and a hypermethylated group (n = 13), and the median expression is displayed here. The tissue of origin of individual cell lines can be seen in Table 1. Figure 2 shows up-regulation of MAL expression after drug treatment. Decreased MAL promoter methylation followed by upregulation of mRNA expression in colorectal cancer cell lines was observed after treatment with demethylated 5-aza-2'-deoxycytidine alone and in combination with the deacetylase inhibitor trichostatin A It was seen. The upper panel shows the relative expression level of MAL in two colon cancer cell lines HT29 and HCT15 treated with 2-aza-2′-deoxycytidine alone, trichostatin A alone, and the two drugs combined (equal scale). Has been demonstrated. The lower panel illustrates MAL MSP results for the same sample. The visible PCR product in lane U indicates the presence of an unmethylated allele, while the lane M PCR product indicates the presence of a methylated allele. Abbreviations: AZA, 5-aza-2′-deoxycytidine; TSA, trichostatin A; Pos, positive control (unmethylated reaction: DNA from normal blood, methylation reaction: methylated DNA in vitro); Neg, ( Negative control with water as template); U, lane for unmethylated MSP product; M, lane for methylated MSP product. Figure 2 shows MAL expression in colorectal cancer. Positive cytoplasmic staining for MAL is seen in renal tubules (A), and no staining is observed in myocardium (B), consistent with previous reports (Marazuela M, et al J Histochem Cytochem 2003, 51: 665-674). Colorectal cancer epithelial cells were MAL negative (C, D), whereas in normal colon tissue, cytoplasmic expression of MAL was found in both epithelium and connective tissue (E, F). All images were recorded using a 40 × lens (400 × magnification).

  The invention will now be described in further detail below.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined:

Epigenetics
Methylation is an epigenetic change defined as a non-sequence-based change that is inherited through cell division.

Methylation “Hypermethylation” in this context is methylation that exceeds standard methylation. Reference methylation is the methylation of a gene in a sample from a healthy subject or normal tissue. Thus, methylated genes that are unmethylated in normal tissues will be classified as hypermethylated. A “methylation state” is the degree to which methyl modifications are present or absent at one or more CpG sites within at least one nucleic acid sequence. It should be understood that the methylation status of one or more CpG sites is preferably measured in multiple copies of a particular gene of interest.

  “Methylation level” is a representation of the amount of methylation in one or more copies of a gene or nucleic acid sequence of interest. The methylation level can be calculated as an absolute measure of methylation within the gene or nucleic acid sequence of interest. The “relative methylation level” is measured as the amount of methylated DNA relative to the total amount of DNA present, or as the number of methylated copies of the gene or nucleic acid sequence of interest relative to the total number of copies of the nucleic acid sequence or genes. Can do. In addition, “methylation level” can be measured as a percentage of methylated CpG sites within the DNA range of interest.

  The term methylation level also covers, for example, situations where one or more CpG sites in the promoter region are methylated, but the amount of methylation is below the amplification threshold. Therefore, the methylation level may be an estimated value of the methylation amount in the gene of interest.

  The present invention is not limited in any way to a specific type of assay for measuring the methylation status or methylation level of a gene according to the present invention.

  In one embodiment, if the methylation level of the gene of interest is 15% to 100%, 50% to 100%, more preferably 60% to 100%, more preferably 70% to 100%, more preferably 80%. % To 100%, more preferably 90% to 100%. Thus, in one embodiment of the invention, the methylation level of the gene according to the invention is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

CpG
A “CpG site” is a DNA region in which a cytosine nucleotide appears next to a guanine nucleotide of a linear base sequence along its length. “CpG” represents cytosine and guanine separated by phosphate, which binds two nucleosides in DNA. The “CpG” notation is used to distinguish between cytosine that forms base pairs with guanine and cytosine followed by guanine.

  A CpG island can be defined as a contiguous window of at least 200 base pairs of DNA with a G: C content of at least 50% and a ratio of the observed CpG frequency divided by the expected frequency exceeding 0.6. However, they also have a G: C content of at least 55% and more than a 500 base pair window with a ratio of the observed CpG frequency of at least 0.65 divided by the expected CpG frequency. Stringent definitions can also be specified.

Promoter region or sequence A “promoter region or sequence” comprises a continuous nucleic acid sequence extending 1000 bp upstream from the transcription start site of a given gene, and a continuous nucleic acid sequence extending 300 base pairs downstream from the transcription start site. In the sequence list, upstream sequences are shown in lower case, while downstream sequences are shown in upper case. In the 3 ′ part of the sequence, intron sequences are shown in lower case.

Transcription Start Site “Transcription start site” is used in conjunction with the present invention to describe the point at which transcription is initiated. Transcription can begin at one or more sites within a gene, and a gene can have multiple transcription initiation sites, some of which can be specific for transcription in a particular cell type or tissue.

Nucleic acid sequence methylation Genes are DNA regions involved in the production and regulation of polypeptide chains. Genes include both coding and non-coding portions including introns, exons, promoters, initiators, enhancers, terminators, microRNAs, and other regulatory elements. As used herein, “gene” means at least part of a gene. Thus, for example, a “gene” can be considered a promoter for the purposes of the present invention. Accordingly, in one embodiment of the invention, at least one member of the panel of genes comprises a non-coding portion of the entire gene. In certain embodiments, the non-coding portion of the gene is a promoter. In other embodiments, all members of the entire panel in the gene comprise non-coding portions of the gene, such as but not limited to introns. In other specific embodiments, the non-coding portion of the member in the gene is a promoter. In other embodiments of the invention, at least one member of a panel within a gene comprises the coding portion of that gene. In other embodiments, all members of the entire panel in the gene comprise the coding portion of the gene.

  The term “nucleic acid sequence” refers to a polymer of deoxyribonucleotides in either single-stranded or double-stranded form.

  A “partial sequence” is any part of the entire sequence. Thus, a partial sequence refers to a contiguous sequence of nucleic acids that are part of an amino acid or longer sequence nucleic acid (eg, a polynucleotide).

  The term “sequence identity” indicates a quantitative measure of the degree of homology between two nucleic acid sequences of equal length. If the two sequences to be compared are not of equal length, they either allow for the insertion of a gap, or alternatively allow for cleavage at the end of the polypeptide or nucleotide sequence, It must be aligned to provide the best fit that can be achieved. The sequence identity is as follows:

{ Where N dif is the total number of residues that are not identical in the two sequences when aligned, and N ref is the number of residues in one sequence. }. Thus, the DNA sequence AGTCAGTC will have 75% sequence identity with the sequence AATCAATC (N dif = 2 and N ref = 8). The gap is considered not identical among the specific residues, ie the DNA sequence AGTGTC will have 75% sequence identity with the DNA sequence AGTCAGTC (N dif = 2 and N ref = 8).

  For all claims of the invention relating to nucleotide sequences, the percentage of sequence identity between one or more sequences can also be calculated using crustalW software (http://www.ebi.ac.uk/clustalW/index) using default settings. .Html). These settings for nucleotide sequence alignment are as follows: Alignment = 3Dfull, Gap Open 10.00, Gap Ext. 0.20, Gap separation Dist. 4. DNA weight matrix: identity (IUB). Alternatively, sequences can be analyzed using the program DNASIS Max, and sequence comparisons can be made at www. paralian. org. This service is based on two comparison algorithms called Smith-Waterman (SW) and ParAlign. The first algorithm was established by Smith and Waterman (1981) and is a well-established method for finding the optimal local alignment of two sequences. The other algorithm, ParAlign, is a heuristic method for sequence alignment; details on that method have been published by Rognes et al. The default settings for score matrix and gap penalties, and E values were used.

  In the context of the present invention, “complementary” refers to the exact pairing capacity between two nucleotide sequences of each other. For example, if a nucleotide at a particular position in an oligonucleotide is capable of hydrogen bonding with a nucleotide at the corresponding position in the DNA molecule, then the oligonucleotide and the DNA are considered to be complementary to each other at that position. . DNA strands are considered to be complementary to each other when a sufficient number of nucleotides in the oligonucleotide can form hydrogen bonds with the corresponding nucleotides in the target DNA, allowing the formation of a stable complex.

  In this context, the expression “complementary sequence” or “complement” therefore also refers to a nucleotide sequence that anneals to a nucleic acid molecule of the invention under stringent conditions.

  The term “stringent conditions” refers to the overall conditions of high, weak or low stringency.

  The term “stringency” is well known in the art and is used in reference to the conditions under which nucleic acid hybridization takes place (temperature, ionic strength, and the presence of other compounds such as organic solvents). “High stringency” conditions will result in nucleobase pairing only between nucleic acid fragments having a high frequency of complementary base sequences compared to “weak” or “low” stringency conditions. Suitable conditions for test hybridization are presoaked in 5 × SSC and 20% formamide, 5 × Denhart solution, 50 mM sodium phosphate, pH 6.8, and 50 mg of denatured sonicated pups. Prehybridization in bovine thymus DNA solution at ˜40 ° C. for 1 hour, followed by hybridization for 18 hours at ˜40 ° C. in the same solution supplemented with 100 mM ATP, followed by 2 × SSC, 0 .2% SDS at 40 ° C. (low stringency), preferably at 50 ° C. (moderate stringency), more preferably at 65 ° C. (high stringency), even more preferably ~ 75 Includes 3 filter washes for 30 minutes at 0 ° C (very high stringency). For further details regarding hybridization methods, see Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor, 1989.

Cancer “Cancer” is a cell that is aggressive (proliferates and divides regardless of normal restrictions), is invasive (invades and destroys surrounding tissues), and sometimes metastatic (in the body). Is a group of diseases that spread to other places). These three malignant features of cancer distinguish them from benign tumors that are self-limiting in growth and usually do not invade or metastasize.

  Cancer is usually classified by the tissue in which the cancer cells originated, as well as the normal cell types to which they are most similar. A definitive diagnosis typically requires histological examination of tissue biopsy material by a pathologist. The prognosis of a cancer patient is most affected by the type of cancer and the stage or extent of the disease. Early diagnosis is usually associated with better treatment and higher survival rates.

  A “tumor suppressor gene” is a gene that is often inactivated in cancer cells and is involved in precise DNA replication, cell cycle control, tissue orientation and adhesion, and interaction with the protective cells of the immune system. It results in a loss of normal function of those cells such as action. In several types of cancer, including colorectal cancer, several tumor suppressor genes have been confirmed to be epigenetically inactivated by hypermethylation of the CpG island promoter.

  A tumor can be an abnormal swelling, lump, or mass, however, because the term is an interpretation herein, the term means a neoplasm, particularly a solid neoplasm. A neoplasm is defined as an abnormal growth of genetically engineered cells. Neoplasms can be benign or malignant. A malignant neoplasm or malignant tumor is here understood as a cancer. A benign neoplasm or benign tumor is a tumor (solid neoplasm) that normally stops growing by itself and does not invade other tissues and does not form metastases. However, benign tumors can become malignant.

  A tumor that invades the surrounding tissue is understood herein as cancer. A pre-malignant tumor, a pre-cancerous condition, or a pre-invasive tumor is understood herein as a neoplasm that has the potential to progress to cancer (becomes invasive) if left untreated but untreated. Is done.

  The method according to the invention can be used to determine the severity, i.e. stages such as the Dukes system, the Astler-Coller system, and the TNM staging AJCC (American Joint Committee on Cancer). The Dukes system is a 4-class staging system that classifies colorectal cancer from A to D based on the extent of the tumor: A, penetration into the intestinal wall, but no penetration; B, penetration through the intestinal wall; C, lymph node metastasis regardless of the extent of intestinal wall penetration; D, spread of cancer to distant organs such as liver and lung. Many variations of this taxonomy exist, for example, TNM staging.

Biomarker A biomarker may be a substance whose detection indicates a specific medical condition. Biomarkers may also indicate changes in protein expression or status that correlate with disease risk or progression, or disease susceptibility to a given treatment. Superior biomarkers can be used to diagnose an individual's disease risk, the presence of the disease, or personalized treatment for an individual's disease. The terms biomarker and marker are used interchangeably in this context.

  Cancer markers, tumor markers, and methylation markers in this context are markers for detecting tumors and / or cancer. A marker is used to detect cancer and / or tumor in a subject, or cancer that develops, and / or the presence of a tumor, or whether a subject is likely to develop or relapse with cancer and / or tumor. Can be used to detect if.

  Each gene according to the present invention may be a marker, biomarker, cancer marker, or tumor marker.

Tumor progression In addition to determining whether a subject has developed, is or is likely to develop cancer, or whether the subject has relapsed after cancer treatment, the method according to the present invention comprises: It can also be used to detect cancer progression in This can be done by measuring the methylation status or level of one or more genes of interest at different time points, and then measuring the difference in the methylation status or level of one or more genes over time. . Differences in methylation status or levels over time can be an indicator of whether a subject has developed, is developing or is likely to develop cancer, or whether the subject has relapsed after cancer treatment.

  The present invention also provides a method for producing a prognosis related to a disease course in a human cancer patient. For the purposes of the present invention, the term “prognosis” is intended to encompass prediction and probabilistic analysis of disease progression, particularly tumor recurrence, metastatic spread, and disease recurrence. The prognostic methods of the present invention are used clinically in making decisions regarding treatment modalities, including therapeutic intervention, diagnostic criteria such as disease staging and disease monitoring, and monitoring metastasis or recurrence of neoplastic disease. Shall be. Treatment is understood herein as prophylactic and curative therapy.

  The present invention also provides a method for confirming the results or signs obtained by the aforementioned methods, such as test or screening methods.

  Thus, the phrase “whether or not cancer has developed, is or is likely to develop, or whether the subject has relapsed after cancer treatment” is used herein to refer to the current presence of cancer, the future Covering decisions and / or predictions such as estimation or determination of the likelihood of a new occurrence or future recurrence.

Sample A sample is, without limitation, a tissue section or biopsy specimen, such as a portion of a neoplasm to be treated, or it may be a portion of surrounding normal tissue. The sample is preferably, but not limited to, blood, stool (feces), urine, pleural fluid, bile, bronchial fluid, oral washings, tissue biopsy, ascites, pus, brain Spinal fluid, punctate, follicular fluid, tissue or mucus. The sample may be processed before being assayed. For example, the sample may be diluted, concentrated, or purified, and / or at least one compound, such as an internal standard, may be added to the sample. Procedures for handling separate samples are known to those skilled in the art. It should be understood that all methods according to the present invention preferably relate to in vitro analysis of samples.

Sample methylation frequency The term “sample methylation frequency” is defined herein as a quantitative measurement of a methylated sample, ie, the relative number of samples in which the gene of interest is methylated. The By way of example, as can be seen from Table 3, the methylation frequency of the sample of CNRIP1, in which 20 out of 20 samples from the colon cell line are methylated, is 100%. The relative amount of sample that is methylated is compared to a reference or cut-off level that is estimated based on sensitivity and specificity of each gene.

Criteria Standards, reference levels or reference values must be established to determine whether a subject has developed, is likely to develop cancer or is likely to develop, or whether the subject has relapsed after cancer treatment . Criteria also include changes in assays and methods, changes in kits, changes in handling, changes associated with each other or marker combinations with other known markers, and other not directly or indirectly related to methylation. It also allows changes to be taken into account.

In the context of the present invention, the term “criteria” relates to a standard associated with quantity, quality, or type that can be compared with other values or characteristics such as a calibration curve.
A reference or reference level is a value or level determined in this connection by measuring parameters (methylation status or methylation level) of both a healthy control population and a population suffering from a known cancer. It should be understood that the cancer population using either a predetermined specificity or a predetermined sensitivity based on an analysis of the relationship between the parameter values of the healthy control population and the cancer patient population and known clinical data Is identified.

  As is widely understood by those skilled in the art, cancer screening methods are a process of comparative decision making. In any decision-making process, a reference value, reference level, or cut-off point based on the subject with cancer or the pathology of interest and / or the subject without cancer or the pathology of interest is required . The reference level (or cut-off point or cut-off level) is the number of subjects who meet the conditions to continue further open diagnostic tests, for all subjects who continue further diagnostic tests, for example, the incidence of cancer, and All subjects who have an average risk factor for onset, a patient-specific risk higher than a specific risk level such as 1/400 or 1: 250 (as defined by the screening of the group or individual subjects) It is established taking into account several criteria, including the decision that blood diagnostic testing should continue, or other criteria known to those skilled in the art.

  The reference level can be adjusted based on a number of criteria, such as, but not limited to, a particular group of individuals tested. As an example, the cut-off level can be set lower in people with immune deficiency and in patients at high risk of developing active disease, while the reference level can be set for other low risk of developing active disease. Higher in the group of healthy people.

  Reference levels vary with respect to the various stages of the disease (eg, benign or malignant tumor), the origin of normal mucus (from a cancer patient versus from someone who does not have cancer), or the origin of blood and feces obtain. In addition, the reference level can be different for subjects who are susceptible to disease or who have relapsed from treatment of the disease.

  The reference level can be customized to accept inherent sensitivity or specificity: if the person skilled in the art desires a test with high sensitivity, the reference level can be set low. If a person skilled in the art seeks a test with high specificity, the reference level can be set high.

  Depending on the prevalence or predictive prevalence of the disease, the reference level is a causal relationship that determines the severity of the disease and whether the patient is positive for the test or negative for the test. Depending, it can be adjusted to obtain as few false positives or as few false negatives as desired.

  The method of measuring methylation, selected portions of the nucleic acid sequence comprising the promoter region of the marker gene, or other parameters will provide other reference values that can be measured according to the teachings herein.

  If symptomatic single patients must be diagnosed or if the test is used to screen a large number of people in a population, the reference levels can be different.

  Reference levels are based on complex methylation status or level measurements of different markers such as, but not limited to, CNRIP1, SPG20, FBN1, SNCA, INA, MAL, ADAMTS1, VIM, SFRP1, and / or SFRP2. be able to. The reference level of the compound can yield other values that can be measured in accordance with the teachings of the present invention.

  The level of methylation is compared to a set of reference data or reference levels, such as a cutoff value, to determine if the subject is at high risk or likelihood of cancer.

Specificity and Sensitivity The sensitivity of any given screening test is the percentage of people with the condition that are correctly identified or diagnosed by that test, for example, all persons with a given condition are positive tests. In this case, the sensitivity is 100%. The specificity of a given screening test is the percentage of people who do not have a disease state that is correctly identified or diagnosed by that test, for example, if a negative test result is obtained for all people who do not have a disease state, There is 100% specificity.

  Thus, the sensitivity is defined as (number of true positive test results) / (number of true positives + number of false negative test results).

  Specificity is defined as (number of true negative results) / (number of true negatives + number of false positive results).

  The gene according to the present application is highly sensitive (the relative amount of the sample comprising a methylated gene of interest from a subject suffering from cancer is high) and highly specific (the methyl of interest from a subject not suffering from cancer). The relative amount of the sample comprising the activating gene is low).

  A good marker for cancer is a gene that is methylated in almost all samples when the subject is afflicted with cancer, and is not methylated in samples from subjects who are not afflicted with cancer. is there.

  The specificity of the method according to the present invention is preferably 70% to 100%, such as 75% to 100%, more preferably 80% to 100%, more preferably 90% to 100%. Thus, in one embodiment of the invention, the specificity of the invention is 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

  The sensitivity of the method according to the invention is preferably 80% to 100%, more preferably 85% to 100%, more preferably 90% to 100%. Thus, in one embodiment of the present invention, the sensitivity of the present invention is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

  It should be understood that the markers according to the invention can be used in combination in the method according to the invention. The use of several markers in combination often enhances the specificity and / or sensitivity of the assay compared to assays involving the use of a single marker. When several markers are used in combination, it can therefore be tolerated that the specificity and sensitivity of each marker is lower than previously specified.

  As an example illustrated in Table 3, the gene CNRIP1 is present in 20 (100%) of 20 of all samples from colorectal cancer cell lines and in 45 (94%) of 48 of adenoma samples. Methylated—that is, this gene has a high sensitivity with a probability of disease detection of 100 and 94% from each sample. The same gene methylation in a sample from normal tissue is 0 out of 21 and therefore there is no chance of detecting false positives for that gene, so no person suffering from all cancers Is highly specific. Samples from normal mucosa from cancer patients showed methylation in 9 out of 21 samples, indicating that cancer was detected away from the tumor.

Receiver operating characteristics The accuracy of a diagnostic test is best described by its receiver operating characteristics (ROC) (in particular Zweig, MH, and Campbell, G., Clin. Chem. 39 (1993) 561-577). checking). The ROC graph is a plot of sensitivity / specificity pairs due to continuous changes in baseline levels across the entire range of observed data.

  The clinical performance of a laboratory test depends on its diagnostic accuracy or ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy assesses the ability of the test to accurately identify two different conditions of the subject being investigated. Such conditions are, for example, health and disease, latent infection or no recent infection versus infection, or benign disease versus malignancy.

  In each case, the ROC plot shows the overlap between the two distributions by plotting the sensitivity versus the specificity for the entire range of 1-decision thresholds. The Y axis is sensitivity, which is calculated completely from the affected subgroup. The x-axis is the false positive fraction or 1-specific, which is calculated completely from the unaffected subgroup.

  By using test results from two different subgroups, the sensitivity and specificity are calculated completely separately, so the ROC plot is independent of the prevalence of the disease in the sample. Each point on the ROC plot represents a sensitivity / specificity pair that corresponds to a particular decision threshold. A test with perfect discrimination (no overlap between the two distributions of results) has a true positive fraction of 1.0 or 100% (complete sensitivity) and a false positive fraction of 0 (complete A ROC plot that penetrates the upper left corner. The theoretical plot of the test without discrimination (identical outcome distribution for the two groups) is a 45 ° diagonal line from the lower left corner to the upper right corner. Most plots fall between these extremes. (If the ROC plot falls completely below the 45 ° diagonal, this is easily corrected by reversing the “positive” criterion from “more” to “less” or vice versa. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test.

  One convenient goal for quantifying the diagnostic accuracy of a laboratory test is to express its execution by a single number. The most common global measure is the area under the ROC plot. By convention, this region is always ≧ 0.5 (otherwise one skilled in the art can override the decision-making principle that makes it so). Values range from 1.0 (complete separation of 2 groups of test values) to 0.5 (no apparent distribution difference between 2 groups of test values). The area depends on the entire plot, not just on a particular part of the plot, such as the point closest to the diagonal or sensitivity with 90% specificity. This is a quantitative, descriptive representation of how close the ROC plot is (area = 1.0).

  The clinical utility of a novel cancer marker gene can be assessed by comparison with or in combination with other markers for a given cancer, eg, the novel cancer markers CNRIP1, SPG20, FBN1, SNCA, INA, and MAL. Clinical utility: for example, without limitation, with established diagnostic tools that measure corresponding or established methylation marker expression levels such as ADAMTS1, VIM, SFRP1, SFRP2, and CRABP1 Evaluation was made by comparison.

Risk assessment and cut-off A positive test cut-off limit must be established to determine whether the subject is at high risk of developing cancer, for example. This cutoff can be established by the laboratory, by the physician, or individually based on each subject.

  Alternatively, the cut point is determined as a value derived from the mean, median, or geometric mean +/− 1 standard deviation or standard deviation of a negative control group (eg, not suffering from cancer). obtain.

  The cutoff limit for positive test results according to the present invention is the methylation state or level at which methylation is an indicator of cancer.

  Another cut-off point may be the amount of CpG sites that need to be methylated for a gene that is determined to be methylated.

  The inventor has successfully identified a new cancer marker. Since the methylation level of the CpG site or the methylation level of the promoter region of the nucleic acid sequence of a gene selected from CNRIP1, SPG20, FBN1, SNCA, INA, and MAL is enhanced in subjects suffering from cancer, these This gene is an effective marker for cancer detection, for example.

  Cut-off points vary based on the specific conditions of the person being tested, such as but not limited to the risk of suffering a disease, occupation, geographical address, or radiation dose.

  Cut-off points include, but are not limited to, age, gender, genetic background (ie, HLA type), acquired or inherited immune dysfunction (eg, HIV infection, diabetes, kidney or liver failure). Patients tested, such as, but not limited to, patients undergoing treatment with immunomodulators such as corticosteroids, chemotherapy, TNF-α blockers, mitotic inhibitors, etc.) Different based on specific conditions.

  Thus, the execution of the determination or adjustment of the cut-off limit will determine the test sensitivity to detect cancer, if present, or its specificity to exclude cancer or disease if below this limit. And the principle is that values above the cut-off point indicate a high risk and values below the cut-off point indicate a low risk.

Expression Several tumor suppressor genes have been confirmed to be inactivated by CpG island promoter methylation. An example is the MLH1 gene, where hypermethylation of a limited number of CpG sites approximately 200 base pairs upstream of the transcription start point is invariably correlated with a lack of gene expression.

  This analysis of cancer cell lines from several tissues suggests that hypermethylation in a limited region near the MAL transcription start site is associated with reduced or lost gene expression. Quantitative gene expression results from colorectal cancer cell lines analyzed before and after epigenetic drug therapy also suggest that this applies to SPG20, INA, and CRNIP1.

  Thus, measuring the methylation status or level of the gene of interest, as well as its expression, enhances the specificity and sensitivity of the method.

Methods The present invention relates to a particular subset of six genes in which genes that are hypermethylated at an unusually high frequency in cancers such as colorectal cancer are selected from the 21 genes previously discussed by Lind et al. Based on the knowledge that it was found in These highly suitable hypermethylation markers include CNRIP1, SPG20, FBN1, SNCA, INA, and MAL. For example, findings regarding MAL are contrary to previous reports where MAL hypermethylation is seen only infrequently in colorectal cancer.

In a first aspect of the invention, a method for determining whether a subject has developed, is developing or is likely to develop cancer, or whether the subject has relapsed after cancer treatment, comprising the following steps:
a) Methylation level of CpG sites, number of CpG sites methylated, or methylation status in the nucleic acid sequence of the promoter region, first exon or intron of at least one gene in a sample obtained from the above-mentioned subject Measuring,
Where the gene is the following:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
FBN1, for example, identified by ensembl gene ID ENSG000000161477, entrez ID 2200;
SNCA, for example as specified by ensemble gene ID ENSG0000013335, entrez ID 6622; and INA, for example, selected from the group consisting of those specified by ensembl gene ID ENSG00000148798, entrez ID 9118.

In one embodiment, the cancer is a tumor, such as a tumor in the (benign or malignant) airway-digestive system.
The method comprises the following steps:
b) compare the methylation level, the number of methylated CpG sites, or the methylation status of the CpG site to the reference; and c) the methylation level, the number of methylated CpG sites, or CpG If the methylation status of the site is higher than the above standard methylation level, the number of methylated CpG sites, or the methylation status of the CpG site, the subject is more likely to develop or develop cancer. Or is likely to develop, or has recurred after cancer treatment, and the methylation level, number of methylated CpG sites, or methylation status of CpG sites is If the subject's methylation level, number of CpG sites methylated, or methylation status of the CpG site is below the standard, the subject is less likely to develop cancer, has not developed cancer, or develops It can further include determining that the likelihood is low or has not recurred after cancer treatment.

In another aspect, the present invention is a method for determining whether a subject has developed, has developed or is likely to develop cancer, or whether the subject has relapsed after cancer treatment, and includes the following: Step:
a) measuring the methylation level of the sample from the subject, the number of methylated CpG sites, or the methylation status of the CpG sites;
b) creating a percentile plot of the methylation level of the at least one gene obtained from a sample from a healthy population, the number of CpG sites methylated, or the methylation status of CpG sites;
c) Methylation level measured in healthy population, number of methylated CpG sites, or methylation status of CpG sites, and methylation level measured in populations suffering from cancer, methylated CpG sites A ROC (Receiver Operating Characteristic) curve based on the number of or CpG site methylation status;
d) selecting the desired combination of sensitivity and specificity from the ROC curve;
e) determining from the percentile plot the methylation level corresponding to the determined or selected specificity, the number of methylated CpG sites, or the methylation status of the CpG sites; and f) at least one of the above mentioned in the sample The methylation level of one gene, the number of methylated CpG sites, or the methylation status of the CpG sites corresponds to the desired combination of sensitivity / specificity described above, the number of methylated CpG sites Or if it is equal to or higher than the methylation status of the CpG site, it is predicted that the subject is likely to suffer from cancer, and the methylation level of the sample, methylated CpG The number of sites, or the methylation status of the CpG site, represents the methylation level, methylated Cp corresponding to the desired sensitivity / specificity combination described above. If the number of G sites or the methylation status of CpG sites is low, the method comprises predicting that the subject is unlikely or not suffering from cancer.

Specifically, the method described above includes the following:
A) a nucleic acid sequence defined by any of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 16;
B) a nucleic acid sequence that is complementary to the sequence defined in A);
C) a partial sequence of the nucleic acid sequence defined in A) or B);
D) The methylation level of a nucleic acid sequence comprising a sequence selected from the group consisting of a nucleic acid sequence that is at least 75% identical to the sequence defined in A), B), or C), a methylated CpG site Or a method comprising measuring the methylation status of a CpG site.

The method described above also has the following:
A) a nucleic acid sequence defined by any of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 16;
B) a nucleic acid sequence that is complementary to the sequence defined in A);
C) a partial sequence of the nucleic acid sequence defined in A) or B);
D) measuring the methylation status of the CpG site in the nucleic acid sequence of an additional gene selected from the group consisting of nucleic acid sequences that are at least 75% identical to the sequence defined in A), B), or C) May be included.

In other embodiments, the methods of the invention comprise measuring the methylation status of CpG sites within the promoter region of MAL. According to this embodiment, the nucleic acid sequence is:
A) a nucleic acid sequence defined by any of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 16;
B) a nucleic acid sequence that is complementary to the sequence defined in A);
C) a partial sequence of the nucleic acid sequence defined in A) or B);
D) A nucleic acid sequence that is at least 75% identical to the sequence defined in A), B), or C).

  Sequence identifiers 1-16 represent the nucleic acid sequences of the genes mentioned above. As those skilled in the art will recognize, it is within the scope of the present invention to analyze the methylation status of CpG sites within these sequences as well as within their complementary sequences. The table below lists the genes according to the invention and the corresponding ID numbers, sequence identifiers and aliases.

  The nucleic acid sequences according to the invention are listed in the sequence listing. Each sequence is in order of reference and in the 5 'to 3' direction: a contiguous sequence of nucleic acid residues (shown in lower case) located within the 1000 bp region upstream of the transcription start site, followed by transcription initiation It comprises a contiguous sequence of nucleic acid residues (shown in capital letters) and an intron sequence of nucleic acid residues (shown in lower case letters) located downstream of the site.

  The method of the invention can be directed to analyzing specific subsequences, as suggested in item C) above. According to these embodiments, the subsequence of C) has a length of at least 8 nucleic acid residues, such as at least 9 nucleic acid residues, at least 10 nucleic acid residues, at least 11 nucleic acid residues, at least 12 nucleic acid residues. At least 13 nucleic acid residues, at least 14 nucleic acid residues, at least 15 nucleic acid residues, at least 20 nucleic acid residues, at least 25 nucleic acid residues, at least 30 nucleic acid residues, at least 35 nucleic acid residues, at least 40 nucleic acid residues, at least There are 45 nucleic acid residues, at least 50 nucleic acid residues, at least as long as 70 nucleic acid residues, or at least as long as 90 nucleic acid residues. It is generally desirable to proceed with the analysis on a particular length sequence to ensure that the method is sufficiently sensitive.

  For practical purposes, it is also desirable to minimize the length of the subsequence undergoing methylation studies in the method of the invention. Therefore, the partial sequence of C) described above has a length of at most 10 nucleic acid residues, for example, at most 13 nucleic acid residues, at most 14 nucleic acid residues, at most 15 nucleic acid residues, at most 20 nucleic acid residues, 25 nucleic acid residues, 30 nucleic acid residues, 35 nucleic acid residues, 40 nucleic acid residues, 45 nucleic acid residues, 50 nucleic acid residues, 70 nucleic acid residues, 70 nucleic acid residues Preferably, it has a length of 90 nucleic acid residues, a maximum length of 110 nucleic acid residues, a maximum length of about 150 nucleic acid residues, or a maximum length of about 200 nucleic acid residues.

  More particularly, the partial sequence of C) has a length of 8 to 200 nucleic acid residues, such as 8 to 150 nucleic acid residues, 8 to 100 nucleic acid residues, 8 to 75 nucleic acid residues, and 8 to 50 nucleic acid residues. Length of 9-200 nucleic acid residues, for example, 9-150 nucleic acid residues, 9-100 nucleic acid residues, 9-75 nucleic acid residues, 9-50 nucleic acid residues, for example, 10-200 nucleic acid residues, 10-150 nucleic acid residues, 10-100 nucleic acid residues, 10-75 nucleic acid residues, 10-50 nucleic acid residues in length, for example, 11-200 nucleic acid residues, 11- 150 nucleic acid residues, 11-100 nucleic acid residues, 11-75 nucleic acid residues, 11-50 nucleic acid residues in length, or, for example, 12-200 nucleic acid residues in length, for example, 12-150 Nucleic acid residues, 12-100 nucleic acid residues, 12-75 nucleic acid residues in length, or, for example, 12 It is desirable to have a length of about 50 nucleic acid residues.

  The promoter regions of the genes according to the invention are listed in the following table:

  For each of the genes mentioned above, the inventor has identified partial sequences that are particularly useful in the methods of the invention. Therefore, with respect to MAL, the partial sequence of C) is the sequence specified by SEQ ID NO: 17 and its complementary sequence, the sequence specified by SEQ ID NO: 18 and its complementary sequence, the sequence specified by SEQ ID NO: 19 and its complementary sequence , A sequence specified by SEQ ID NO: 20 and its complementary sequence, and a group of sequences consisting of a partial sequence of any of these sequences.

  Regarding the fibrillin 1 gene, the partial sequence of C) is preferably the sequence specified by SEQ ID NO: 22 or its complementary sequence, or one partial sequence thereof.

  With respect to chromosome 2 open reading frame 32, (CNRIP1), the partial sequence of C) is preferably the sequence specified by SEQ ID NO: 23 or its complementary sequence, or a partial sequence of one of these.

  For spastic paraplegia 20, spartin (Troyer syndrome), the partial sequence of C) is preferably the sequence specified by SEQ ID NO: 25 or its complementary sequence, or a partial sequence of one of these.

  For synuclein, alpha (a non-A4 component of the amyloid precursor), the partial sequence of C) is preferably the sequence specified by SEQ ID NO: 29 and its complementary sequence, the sequence specified by SEQ ID NO: 30 and its complementary sequence, And a group of sequences consisting of a partial sequence of any of these sequences.

  With respect to the internexin neuron intermediate fiber protein, alpha, the partial sequence of C) is preferably the sequence specified by SEQ ID NO: 32 or its complementary sequence, or one partial sequence thereof.

  What is useful in the present invention is also not limited to the following: muscle cell enhancer factor 2C (SEQ ID NO: 24), C3orf14 / 14HT021 (SEQ ID NO: 21), ubiquitin protein ligase E3A (SEQ ID NO: 26, 27 and 28), an additional gene selected from the group consisting of X-linked 1 (SEQ ID NO: 31), or a complementary sequence thereof, or a partial sequence thereof, expressed by the brain Or a nucleic acid comprising the promoter region of

Only a few genes so far have been found useful for the early detection of cancer based on promoter region methylation events. However, it is within the scope of the present invention to include in the method an analysis of methylation status or level in the promoter region of known markers for hypermethylation in cancer. Therefore, in a further aspect of the invention, the method comprises the step of measuring the methylation status or level of CpG sites in the promoter region / sequence of one or more genes, wherein the one or more genes described above are below:
Adam metallopeptidase with thrombospondin type 1 motif (ADAMTS1, C3-C5, KIAA1346, METH1);
Vimentin (VIM);
Secreted Frizzled-related protein 1 (SFRP1); and secreted Frizzled-related protein 2 (SFRP2),
Selected from the group consisting of

The promoter regions of these genes are represented by sequence identifiers 35-38. Thus, the method of the present invention comprises the following:
i) a nucleic acid sequence defined by any of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36;
ii) a nucleic acid sequence complementary to the sequence defined in i);
iii) a partial sequence of the nucleic acid sequence defined in i) or ii);
iv) at least 75% identical to the sequence defined in i), ii), or iii), eg, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or Measuring the methylation status of CpG sites within a nucleic acid sequence comprising a sequence selected from the group consisting of at least 99% identical nucleic acid sequences may be included.

  One skilled in the art will further recognize that various promoter regions exhibit some degree of degeneracy. Thus, as indicated under item D) above, the promoter sequence for any particular gene may be a sequence that is not completely identical to the one sequence represented by the sequence identifiers 1-16. . In certain embodiments, the nucleic acid sequence of D) is at least 80% identical to the sequence defined in A), B), or C), eg, defined in A), B), or C). At least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or for example, at least 99.5% identical to the sequences.

  Since the specificity and sensitivity of the gene according to the present invention is very high, each gene may be included in the method of the present invention.

In one embodiment, the method comprises the following:
I) a nucleic acid sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 1-4, and related sequences thereof, and II) among one, two, or three nucleic acid sequences as defined in the previous paragraph Measuring the level of methylation, the number of methylated CpG sites, or the methylation status of the CpG sites in the nucleic acid sequence within the promoter region.

In one embodiment, the method comprises the following:
I) a nucleic acid sequence comprising the sequence consisting of SEQ ID NO: 6 and its related sequences, and II) a nucleic acid sequence within the promoter region in one, two or three nucleic acid sequences as defined in the previous paragraph Measuring the level of methylation, the number of CpG sites methylated, or the methylation status of CpG sites.

In one embodiment, the method comprises the following:
I) a nucleic acid sequence comprising a sequence consisting of SEQ ID NO: 7, and its related sequences, and II) a nucleic acid sequence within a promoter region within one, two or three nucleic acid sequences as defined in the previous paragraph Measuring the level of methylation, the number of CpG sites methylated, or the methylation status of CpG sites.

In one embodiment, the method comprises the following:
I) a nucleic acid sequence comprising the sequence consisting of SEQ ID NO: 9 and its related sequences, and II) a nucleic acid sequence within the promoter region in one, two or three nucleic acid sequences as defined in the previous paragraph Measuring the level of methylation, the number of CpG sites methylated, or the methylation status of CpG sites.

In one embodiment, the method comprises the following:
I) a nucleic acid sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 13-14, and related sequences thereof, and II) among one, two, or three nucleic acid sequences as defined in the previous paragraph Measuring the level of methylation, the number of methylated CpG sites, or the methylation status of the CpG sites in the nucleic acid sequence within the promoter region.

In one embodiment, the method comprises the following:
I) a nucleic acid sequence comprising a sequence consisting of SEQ ID NO: 16, and its related sequences, and II) a nucleic acid sequence within a promoter region within one, two or three nucleic acid sequences as defined in the previous paragraph Measuring the level of methylation, the number of CpG sites methylated, or the methylation status of CpG sites.

  The method according to the invention is aimed at detecting or diagnosing cancers such as tumors within the airway-digestive system. “Airway-digestive system” or “airway-gastrointestinal tract” includes lung and gastrointestinal tract: esophageus, stomach, pancreas, liver, gallbladder / bile duct, small intestine, and large intestine including colon and rectum . Specifically, tumors include: colorectal tumors, lung tumors (including small cell lung cancer and / or non-small cell lung cancer), esophageal tumors, stomach tumors, pancreatic tumors, liver tumors It can be selected from the group consisting of tumors, gallbladder and / or bile duct tumors, small intestine tumors, and large intestine tumors.

  Thus, in one embodiment of the invention, the cancer is: colorectal tumor, lung tumor (including small cell lung cancer and / or non-small cell lung cancer), esophageal tumor, stomach tumor, pancreas Tumor, liver tumor, gallbladder and / or bile duct tumor, small intestine tumor, and large intestine tumor.

  In order to determine the number of methylated CpG sites, methylation status of CpG sites, or methylation levels within the aforementioned promoter regions / sequences, the method according to the present invention is sufficient to Requires an amount of DNA. Those skilled in the art will know suitable techniques for isolating and purifying DNA in the required quantity and quality. For most purposes, DNA can be isolated from blood samples, stool samples, tissue samples, or mucus samples from the lungs from the aforementioned subjects. In general, whenever this is possible, it is desirable to carry out the method according to the invention in a non-invasive manner: for gastrointestinal cancer, it is often practical to recover DNA from stool samples And simple. In connection with lung tumors, DNA is isolated from a sample of mucus from the lung, but can provide a convenient approach to non-invasive collection of DNA. For other tumors, including liver and pancreatic tumors, it is preferred to obtain tissue samples for subsequent DNA separation.

  Thus, in one embodiment, the sample is obtained from blood, stool, urine, pleural effusion, bile, bronchial fluid, gargle, tissue biopsy, ascites, pus, cerebrospinal fluid, puncture fluid, follicular fluid, tissue, or mucus. It is done.

  When the method according to the invention is used solely for the purpose of determining the presence of an airway-digestive system cancer or tumor, in particular, a “yes / no” type result is sought there. It is desirable if the method can be limited to analysis of methylation levels or methylation status of CpG sites within the promoter region of 2-4 genes. This clearly requires that the gene has a very high frequency of hypermethylation during the development and progression of cancer.

  Thus, for simple diagnostic purposes, it is most preferred to limit the analysis of promoter regions within only a few genes. As mentioned earlier, this requires the availability of a panel of markers for hypermethylation, where each marker has a high sensitivity and specificity. However, for other more subtle purposes, it may be necessary to analyze the methylation status or level of the promoter region within more marker genes. The method according to the invention therefore comprises at least two genes, including at least one gene as defined in claim 1, in order to determine the risk level of tumor development and / or progression of a subject, About 3 genes, at least about 4 genes, at least about 5 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, At least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, or at least Promoter of about 20 genes It may include the step of measuring the methylation status of CpG sites within the nucleic acid sequence of the region / sequence.

  In accordance with what has been described above, the method of the present invention comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10 types, at least 11 types, at least 12 types, at least 13 types, at least 14 types, at least 15 types, at least 16 types, at least 17 types, at least 18 types, at least about 19 types, or at least about 20 types. And further measuring the methylation status of CpG sites in the additional nucleic acid sequences or their related sequences.

  Thus, for most purposes, it would not be sufficient to analyze the methylation level of the CpG site, the number of methylated CpG sites, or the methylation status of the promoter region of a single gene. . The method according to the invention therefore comprises at least two genes, at least about three genes, at least about four genes, at least one gene selected from the group consisting of the previously defined genes, About 5 genes, at least about 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least Nucleic acid sequences of promoter regions / sequences of 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, or at least about 20 genes CpG site in It may include the step of measuring the methylation status.

  Thus, in another aspect, the invention relates to a method in which the methylation level of at least one additional marker, the number of methylated CpG sites, or the methylation status of CpG sites is measured.

Here, at least one additional marker is:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
FBN1, for example, identified by ensembl gene ID ENSG000000161477, entrez ID 2200;
SNCA, eg, identified by ensembl gene ID ENSG0000013335, entrez ID 6622; and INA, eg, identified by ensembl gene ID ENSG00000148798, entrez ID 9118;
MAL, for example selected from the group consisting of those specified by the ensembl gene ID ENSG000000172005.

Marker gene combinations Thus, for reasons explained above, methylation levels or methylation status can be combined with measurements of one or more other markers and compared to a composite reference level. The measured marker levels can be combined by arithmetic operations such as addition, subtraction, and multiplication, as well as percentage, square root, power, and logarithmic calculation operations. The levels can also be combined according to operations using various models, such as logistic regression and maximum likelihood estimation. Various biomarker combinations and various means of calculating the combined reference value can be implemented by means known to skilled addressees.

  Thus, another aspect of the invention relates to a method according to any of the foregoing claims, wherein the methylation level or methylation status of at least one additional marker is measured.

  The at least one additional marker may be CNRIP1, SPG20, FBN1, SNCA, INA, MAL, ADAMTS1, VIM, SFRP1, or SFRP2, and CRABP1, without being limited thereto. The marker can be compared to a set of reference data to determine whether the subject has cancer or is at high risk of developing cancer.

  A method for constructing a diagnostic test based on a composite marker can be achieved by combining methylation levels or methylation states (or values derived therefrom) of two or more individual markers by a calculation operation (eg, addition). Since there is diversity in the methylation level or methylation status of different markers, for example, it is relevant to compare measurements so that combinations are achieved regardless of differences in methylation level or situation. ing. This can be done by simple normalization from the median or average from the standard.

Synergistic effect A synergistic effect can be achieved by the combination of different marker genes according to the invention.
Specifically, as used herein, a synergistic effect is that for a number of markers acting together, for diagnostic purposes, compared to those predicted by knowing only the sensitivity or specificity of separate markers. Refers to a phenomenon that produces a “composite marker signal” with higher sensitivity or specificity.

  Thus, in one aspect of the invention, the combination of at least one additional marker (eg, CNRIP1, SPG20, FBN1, SNCA, INA, MAL) provides a synergistic effect on sensitivity and / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1 and INA provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1 and SNCA provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1 and FBN1 provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1 and SPG20 provides a synergistic effect with respect to sensitivity / or specificity.

  In another embodiment of the invention, the combination of the markers INA and SNCA provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers INA and FBN1 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers INA and SPG20 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers SNCA and FBN1 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers SNCA and SPG20 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers FBN1 and SPG20 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1 and MAL provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers SPG20 and MAL provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers FBN1 and MAL provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers SNCA and MAL provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers INA and MAL provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, SPG20, and INA provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, SPG20, and FBN1 provides a synergistic effect on sensitivity / or specificity.

  In other aspects of the invention, the combination of the markers CNRIP1, SPG20, and SNCA provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, INA, and SNCA provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, INA and FBN1 provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, SNCA, and FBN1 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers SNCA, SPG20, and FBN1 provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers INA, SPG20, and FBN1 provides a synergistic effect on sensitivity / or specificity.

  In other aspects of the invention, the combination of the markers INA, SNCA, and FBN1 provides a synergistic effect on sensitivity / or specificity.

  In other embodiments of the invention, the combination of the markers INA, SPG20, and SNCA provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers MAL, SPG20, and SNCA provides a synergistic effect with respect to sensitivity / or specificity.

  In other embodiments of the invention, the combination of the markers MAL, INA, and SNCA provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers MAL, INA, and SPG20 provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers MAL, FBN1, and SNCA provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers MAL, FBN1, and SPG20 provides a synergistic effect on sensitivity / or specificity.

  In other aspects of the invention, the combination of the markers MAL, FBN1, and INA provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers MAL, FBN1, and CNRIP1 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers MAL, CNRIP1, and SNCA provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers MAL, CNRIP1, and SPG20 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers MAL, CNRIP1, and INA provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, FBN1, SNCA, and INA provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers SPG20, FBN1, SNCA, and INA provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, SNCA, INA, and SPG20 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, FBN1, INA, and SPG20 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, FBN1, SNCA, and SPG20 provides a synergistic effect on sensitivity / or specificity.

  In other aspects of the invention, the combination of the markers MAL, FBN1, SNCA, and SPG20 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, MAL, SNCA, and SPG20 provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, FBN1, MAL, and SPG20 provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, FBN1, SNCA, and MAL provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers INA, FBN1, SNCA, and MAL provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, INA, SNCA, and MAL provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, FBN1, INA, and MAL provides a synergistic effect with respect to sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, SPG20, FBN1, SNCA, and INA provides a synergistic effect on sensitivity / or specificity.

  In other aspects of the invention, the combination of the markers MAL, SPG20, FBN1, SNCA, and INA provides a synergistic effect with respect to sensitivity / or specificity.

  In other aspects of the invention, the combination of the markers CNRIP1, MAL, FBN1, SNCA, and INA provides a synergistic effect on sensitivity / or specificity.

  In another aspect of the invention, the combination of the markers CNRIP1, SPG20, MAL, SNCA, and INA provides a synergistic effect with respect to sensitivity / or specificity.

  In other aspects of the invention, the combination of the markers CNRIP1, SPG20, FBN1, MAL, and INA provides a synergistic effect on sensitivity / or specificity.

  In other aspects of the invention, the combination of the markers CNRIP1, SPG20, FBN1, SNCA, and MAL provides a synergistic effect on sensitivity / or specificity.

Thus, in one embodiment, the method according to the invention comprises the following:
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
FBN1, for example, identified by ensembl gene ID ENSG000000161477, entrez ID 2200;
At least one additional marker selected from the group consisting of SNCA, eg, identified by ensembl gene ID ENSG0000013335, entrez ID 6622; and INA, eg, identified by ensembl gene ID ENSG00000148798, entrez ID 9118 Combined with the step of measuring methylation level, number of methylated CpG sites, or methylation status of CpG sites, methylation level of CNRIP1, number of methylated CpG sites, or methylation status of CpG sites Measuring.

Thus, in one embodiment, the method according to the invention comprises the following:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
FBN1, for example, identified by ensembl gene ID ENSG000000161477, entrez ID 2200;
At least one additional marker selected from the group consisting of SNCA, eg, identified by ensembl gene ID ENSG0000013335, entrez ID 6622; Combined with the step of measuring methylation level, number of methylated CpG sites, or methylation status of CpG sites, methylation level of SPG20, number of methylated CpG sites, or methylation status of CpG sites Measuring.

Thus, in one embodiment, the method according to the invention comprises the following:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
At least one additional marker selected from the group consisting of SNCA, eg, identified by ensembl gene ID ENSG0000013335, entrez ID 6622; Combined with the step of measuring methylation level, number of methylated CpG sites, or methylation status of CpG sites, methylation level of FBN1, number of methylated CpG sites, or methylation status of CpG sites Measuring.

Thus, in one embodiment, the method according to the invention comprises the following:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
At least one additional marker selected from the group consisting of FBN1, eg, identified by ensemble gene ID ENSG000000161477, entrez ID 2200; Combined with the step of measuring methylation level, number of methylated CpG sites, or methylation status of CpG sites, the methylation level of SNCA, the number of methylated CpG sites, or the methylation status of CpG sites Measuring.

Thus, in one embodiment, the method according to the invention comprises the following:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
At least one additional marker selected from the group consisting of FBN1, eg, identified by ensemble gene ID ENSG00001614747, entrez ID 2200; In combination with the step of measuring methylation level, number of methylated CpG sites, or methylation status of CpG sites, methylation level of INA, number of methylated CpG sites, or methylation status of CpG sites Measuring.

Bisulfite treatment and methylation specific polymerase chain reaction The present invention is not limited by the type of assay used to assess the methylation status of a gene or member of a gene panel. In fact, any assay used to measure the methylation status or level of a gene or gene panel should be sufficient for the purposes of the present invention.

  An actual approach to measure the methylation status of CpG islands within the promoter region may involve treating the promoter sequence with bisulfite. Bisulfite treatment of DNA causes sequence variation as unmethylated, but methylated cytosine is not converted to uracil. Bisulfite treatment followed by sequence analysis reveals that 5-methyl cytosine appears in the final sequence as unmethylated cytosine, while 5-methyl cytosine appears as cytosine in the gene promoter after bisulfite modification. Allows positive display of methyl cytosine. In a particular embodiment of the invention, the methylation status of the aforementioned promoter region / sequence is therefore measured by nucleic acid sequencing (bisulfite sequencing).

  In a further aspect of the invention, the number of methylated CpG sites, methylation status of CpG sites, or methylation level of the aforementioned promoter region / sequence is measured by methylation specific PCR. In the examples for this application, a set of suitable PCR conditions and primer designs are given. In general, however, those skilled in the art will have the necessary knowledge to be able to determine the appropriate conditions and primer design for PCR analysis.

  As those skilled in the art know, real-time fluorescence provides a simple and rapid approach to the detection of PCR products, and it can be easily applied to diagnostic procedures that require high-throughput processing. In a presently preferred embodiment of the invention, said methylation specific PCR thus comprises real-time fluorescent detection of the PCR product.

  For most other PCR procedures, the method of the invention can include a step of separating the products according to size. Specifically, the method of the present invention can comprise a step of separating PCR products obtained by gel or capillary electrophoresis.

  As part of the analysis, the resulting PCR product can be detected by use of a label selected from the group consisting of a fluorescent label, a chemiluminescent label, and a radioactive label. For safety and practical reasons, non-radioactive labels are preferred for most purposes.

  The methylation status or level of the aforementioned promoter regions / sequences can also be measured by pyrosequencing, by mass spectrometry, or by the use of methylation specific restriction enzymes.

  The methylation level, the number of methylated CpG sites, or the methylation status of CpG sites is not limited to this, but bisulfite sequencing, quantitative, and / or qualitative methylation specific Polymerase chain reaction (MSP), pyrosequencing, Southern blotting, restriction enzyme landmark genome scanning (RLGS), single nucleotide primer extension method, CpG island microarray, SNUPE, COBRA, mass spectrometry, methylation specific This is measured by using a typical restriction enzyme, measuring the expression level of the aforementioned gene, or a combination thereof.

  In a preferred embodiment, the methylation-specific PCR used in the methods of the invention uses nucleic acid primers that can hybridize to a nucleic acid sequence comprising two CpG sites and a cytosine residue that is not within the CpG site. Comprising. Inclusion of such cytosine residues that are not methylated is desirable to better distinguish between non-bisulfite converted DNA and bisulfite converted DNA. The primer for the methylated sequence will still bind to the methylated CpG site, which is the site that remains CpG after bisulfite conversion. In the presence of unaltered DNA, this will include a CpG site regardless of the methylation status, and then methylation specific primers bind to the unaltered DNA and produce false positives. right. Inclusion of a “C” that is not in the CpG site within the region targeted by the primer contains a “T” at the same site while the converted DNA contains a “C”, so that the primer Prevents binding to unconverted DNA.

  In an even further embodiment, methylation specific PCR comprises the use of a nucleic acid primer capable of hybridizing to a nucleic acid sequence comprising two CpG sites and a cytosine residue not within the CpG site.

  The method according to the invention can also be combined with other known parameters relating to cancer. Thus, the methylation level or status of the gene of the present invention is not limited thereto, but includes the following parameters relating to cancer: gene DNA integrity assay, ploidy, gene mutation status, genomic variation, fusion It can be combined with any of genes, splicing mutations, expression differences, miRNAs.

Use The markers according to the present invention are very suitable for use as cancer markers due to their high sensitivity and specificity. Thus, another aspect of the present invention is methylation as an indicator as to whether a subject has developed, is developing, is likely to develop cancer, or whether the subject has relapsed after cancer treatment. In a diagnostic assay in which the level, number of methylated CpG sites, or methylation status of CpG sites is assessed:
CNRIP1, eg, identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
FBN1, for example, identified by ensembl gene ID ENSG000000161477, entrez ID 2200;
Use of one or more genes selected from the group consisting of SNCA, eg, identified by ensemble gene ID ENSG0000013335, entrez ID 6622; Related to.

Other aspects are methylated levels, methylated as an indicator of whether the subject has developed, is likely to develop cancer, is likely to develop cancer, or whether the subject has relapsed after cancer treatment Said nucleic acid in a diagnostic assay in which the number of CpG sites or the methylation status of CpG sites is assessed is:
A) a nucleic acid sequence defined by any of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 16;
B) a nucleic acid sequence complementary to the sequence defined in A);
C) a partial sequence of the nucleic acid sequence defined in A) or B);
D) relates to the use of a nucleic acid sequence comprising a nucleic acid sequence selected from the group consisting of nucleic acid sequences that are at least 75% identical to the sequence defined in A), B) or C).

Antibodies The present invention also relates to antibodies against methylated sequences. Thus, other aspects of the invention include the following:
A) a nucleic acid sequence defined by any of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 16;
B) a nucleic acid sequence complementary to the sequence defined in A);
C) a partial sequence of the nucleic acid sequence defined in A) or B);
D) relates to an antibody that recognizes a methylated nucleic acid sequence selected from the group consisting of nucleic acid sequences that are at least 75% identical to the sequence defined in A), B) or C).

Diagnostic Kit In a preferred aspect, the present invention includes the following:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
FBN1, eg, identified by 2200 with ensembl gene ID ENSG00000166147, entrez ID;
Is complementary to the nucleic acid sequence of a gene selected from SNCA, eg, ensemble gene ID ENSG0000013335, entrez ID 6622; and INA, eg, ensemb gene ID ENSG00000148798, specified by entrez ID 9118, Diagnostic kits for determining cancer comprising one or more oligonucleotide primers or one or more sets of oligonucleotide primers are provided.

The second aspect of the invention is that each one or more oligonucleotide primers or one or more sets of oligos that are complementary / hybridized to a nucleic acid sequence within the promoter region / sequence of one or more genes Nucleotide primer, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more A diagnostic kit comprising about 17 or more, 18 or more, about 19 or more, or about 20 or more oligonucleotide primers or sets of oligonucleotide primers, wherein the one or more genes described above are:
CNRIP1, for example, those identified by ensembl gene ID ENSG00000119865, entrez ID 25927;
SPG20, for example, identified by ensemble gene ID ENSG0000013104, entrez ID 23111;
FBN1, eg, identified by 2200 with ensembl gene ID ENSG00000166147, entrez ID;
The diagnostic kit is selected from the group consisting of SNCA, eg, identified by ensembl gene ID ENSG0000013335, entrez ID 6622; and INA, eg, ensemb gene ID ENSG000000148798, identified by entrez ID 9118.

Specifically, the kits according to this aspect of the invention each have the following:
A) a nucleic acid sequence defined by any of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 16;
B) a nucleic acid sequence complementary to the sequence defined in A);
C) a partial sequence of the nucleic acid sequence defined in A) or B);
D) Complementary / hybridizes to a nucleic acid sequence comprising a sequence selected from the group consisting of nucleic acid sequences that are at least 75% identical to the sequence defined in A), B), or C) One or more oligonucleotide primers or a set of oligonucleotide primers that can be.

In certain embodiments, each of the kits is:
Adam metallopeptidase with thrombospondin type 1 motif (ADAMTS1, C3-C5, KIAA1346, METH1);
Vimentin (VIM);
Secreted Frizzled-related protein 1 (SFRP1); and secreted Frizzled-related protein 2 (SFRP2),
MAL (T cell differentiation protein);

Chromosome 3 open reading frame (C3orf14 / 14HT021);
Ubiquitin protein ligase E3A (UBE3A, AS, ANCR, E6-AP, FLJ26981);
X-linked 1 (BEX1) expressed in the brain;
Muscle cell enhancer factor 2C, MEF2c
Comprising one or more oligonucleotide primers or one or more sets of oligonucleotide primers that are complementary / hybridizable to a nucleic acid sequence within a promoter region / sequence of a gene selected from the group consisting of .

In another aspect of the invention, the kits are each:
A) Nucleic acid defined by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 15. Sequence;
B) a nucleic acid sequence complementary to the sequence defined in A);
C) a partial sequence of the nucleic acid sequence defined in A) or B);
D) Complementary / hybridizes to a nucleic acid sequence comprising a sequence selected from the group consisting of nucleic acid sequences that are at least 75% identical to the sequence defined in A), B), or C) One or more oligonucleotide primers or a set of oligonucleotide primers that can be.

According to these embodiments, the kits are each of the following:
i) a nucleic acid sequence defined by any of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36;
ii) a nucleic acid sequence complementary to the sequence defined in i);
iii) a partial sequence of the nucleic acid sequence defined in i) or ii);
iv) at least 75% identical to the sequence defined in i), ii), or iii), eg, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least One or more oligonucleotide primers or one or more sets of oligonucleotides that are complementary / hybridized to a nucleic acid sequence comprising a sequence selected from the group consisting of nucleic acid sequences that are as much as 99% identical Comprising a primer.

Considering the very high specificity of MAL as a marker for cancer, the kits are each of the following:
A) a nucleic acid sequence defined by any of SEQ ID NO: 1;
B) a nucleic acid sequence complementary to the sequence defined in A);
C) a partial sequence of the nucleic acid sequence defined in A) or B);
D) one that is complementary / hybridized to a nucleic acid sequence selected from the group consisting of nucleic acid sequences that are at least 75% identical to the sequence defined in A), B), or C) It comprises the above oligonucleotide primers or one or more sets of oligonucleotide primers.

  Various designs can be envisioned for the kit of the present invention. In one embodiment, each of the aforementioned primers or primer sets is in a separate container. This will allow the end user of the kit to prepare different primer mixtures for different purposes. However, according to other embodiments, the primer or set of primers can be supplied in a mixture.

  For certain uses, such as traditional diagnostic purposes, a diagnostic kit can include a small number of primers or sets of primers. Specifically, this requires a “yes / no” type result, for example, where the kit is simply used to determine whether a tumor or carcinoma has developed in the airway-digestive system. This is relevant when the kit is used in a given application. For such purposes, the number of primers or primer sets in the kit can be limited to 2-4, where at least one primer or primer set, eg, at least 2, at least 3, Or at least four primers or sets of primers to the nucleic acid sequence according to SEQ ID NO: 1 to 16, or a sequence that is complementary or partially identical to them as defined in the preceding items C) and D) Complementary / hybridize.

  As discussed above, however, in connection with the method of the present invention, the methylation status of the CpG island of the marker gene of the present invention is also used to determine the level of risk for the development and / or progression of the subject's tumor. Can be used for more complex analyses.

  In such embodiments, the diagnostic kits of the invention will typically include a primer or set of primers that can target more marker genes. Kits for such purposes typically need to contain 5 or more primers or sets of primers, where at least one primer or set of primers, eg, at least 2, at least 3, At least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least about 15, or at least about 16 primers or sets of primers are markers according to the invention It can be complementary / hybridized to the nucleic acid sequence of the gene.

  The diagnostic kit according to the present invention can be used, for example, for PCR analysis (eg, specific polymerase chain reaction (MSP) sequence analysis, bisulfite treatment, bisulfate sequencing, electrophoresis, pyrosequencing, mass spectrometry). Methods, sequence analysis by restriction digest, etc.), quantitative and / or qualitative methylation (eg, pyrosequencing, Southern blotting, restriction enzyme landmark genome scanning (RLGS), single nucleotide primer) Required to perform the necessary analysis (e.g. extension method, CpG island microarray, SNUPE, COBRA, mass spectrometry, use of methylation specific restriction enzymes, or measurement of the expression level of the aforementioned genes) Any reagent or medium may further be included. Specifically, the kit includes: deoxyribonucleoside triphosphate, buffer, stabilizer, thermostable DNA polymerase, restriction endonuclease (including methylation specific endonuclease), and (fluorescent label, chemiluminescent label) And one or more components selected from the group consisting of labels (including radioactive labels). The diagnostic assay according to the present invention may further comprise one or more reagents necessary for the separation of DNA.

  It should be noted that the aspects and features described in connection with one of the aspects of the invention also apply to other aspects of the invention.

  When an object according to the invention, or one of its properties or characteristics, is referred to in singular, it also refers to the object, its characteristics or characteristics in plural. By way of example, when referring to “a polypeptide”, it should be understood that it refers to one or more polypeptides.

  Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” are used to refer to the stated elements, integers, or steps. Or the inclusion of a group of elements, integers, or steps, but it is understood that it does not mean the exclusion of any other element, integer, or step, or group of elements, integers, or steps. Let's go.

  All patent documents and non-patent documents cited in this application are incorporated herein in their entirety.

  The invention will now be described in greater detail in the following non-limiting examples.

Example 1:
Bisulfite treatment and methylation specific PCR
DNA from cell lines and colorectal cancer was treated with bisulfite as previously described (Grunau et al. And Fraga et al.). In contrast, DNA from adenomas was bisulfite treated (Smith-Sorensen et al.) According to the protocol of the CpGenome ™ DNA modification kit (Intergen, Boston, Mass.). Promoter methylation status of MAL, C3orf14, FBN1, MEF2c, CNRIP1, SPG20, UBE3A, SNCA, BEX, and INA, followed by a method that allows discrimination between unmethylated and methylated alleles Analyzed by certain methylation specific PCR (MSP) (Herman et al. And Derks et al.). All primers were designed using MethPrimer (Li and Dahiya) or Methyl Primer Express (Applied Biosystems). Their sequences are listed in Table 1 along with the product fragment length, primer position, and annealing temperature for each PCR method. The fragment was amplified using HotStarTaq DNA Polymerase (QIAGEN Inc., Valencia, Calif.) And all results were confirmed at an independent second week of MSP.

Bisulfite sequencing All fragments were amplified using HotStarTaq DNA Polymerase and eluted from a 2% agarose gel with a MinElute gel extraction kit (QIAGEN). Subsequently, samples were sequenced using a 3730 sequencer (Applied Biosystems) using dGTP BigDye Terminator Cycle Sequencing Ready Kit (Applied Biosystems, Foster City, Calif.). Estimate the amount of methyl cytosine at each CpG site of the various fragments by adding the cytosine signal peak height and the cytosine and thymine signal peak heights as previously described by Melki et al. Calculated by comparison.

  When analyzed by methylation-specific polymerase chain reaction (MSP) analysis, MAL hypermethylation was found to be malignant (83%, 40 of 48 carcinomas) as well as benign colon tumors (73%, 59 adenomas). (43 of them) were observed at a very high frequency (FIG. 1).

Example 2:
Methylation specific polymerase chain reaction (MSP) was performed for genes: MAL, C3orf14, FBN1, SPG20, SNCA, BEX1, INA, CNRIP1, UBE3A, MEF2C.

For each sample (colorectal cancer cell line, colorectal cancer, adenoma, and normal mucosa), l. 3 ug of DNA was bisulfite treated using an EpiTect bisulfite kit (Qiagen Inc., Valencia, Calif.) According to the manufacturer's protocol. The modified DNA was eluted with 40 ul of elution buffer (included in the kit). Since bisulfite modification leads to sequence differences, each gene was amplified using two sets of primers, one specific for the unmethylated template and the other specific for the methylated template ( See the primer list of Example 1). 25 μl of PCR mix includes 1 × PCR buffer, 0.75 ul of bisulfite treated template, 1.5-2.0 mM MgCl 2 , 20 pmol of each primer, 200 μM dNTP, and 0.625 to 1 U of HotStarTaq DNA Polymerase (Qiagen) was included. Human placenta DNA (Sigma Chemical Co, St. Louis, MO, USA) treated with SssI methyltransferase (New England Biolabs Inc., Beverly, MA, USA) in vitro used as a positive control for methylated MSP reaction In contrast, DNA from normal lymphocytes was used as a positive control for unmethylated alleles. Water was used as a negative PCR control for both reactions.

  The PCR program consists of 15 cycles of denaturation at 95 ° C. followed by 35 cycles of 95 ° C. for 30 seconds, annealing temperature for 30 seconds, and 72 ° C. for 30 seconds. The final extension was performed at 72 ° C. for 7 minutes.

Annealing temperature and MgCl 2 concentration for each gene tested so far:
MAL: 56 ° C., 1.5 mM MgCl 2 ;
C3orf14: 53 ° C., 1.5 mM MgCl 2 ;
FBN1: 48 ° C., 1.7 mM MgCl 2 for unmethylation reaction, and 2.0 mM MgCl 2 for methylation reaction;
SPG20: 56 ° C., 1.5 mM MgCl 2 ;
SNCA: 53 ° C., 1.5 mM MgCl 2 ;
BEX1: 51 ° C., 1.5 mM MgCl 2 ;
INA: 55 ° C., 1.5 mM MgCl 2 ;
CNRIP1: 52 ℃, 1.5mM of MgCl 2.

  Results: Methylation of MAL, UBE3A, MEF2C, FBN1, C3orf14, BEX1, INA, SNCA, SPG20, and CNRIP1

  The results obtained by methylation specific polymerase chain reaction (MSP) are presented in Table 3 below:

  The marker genes analyzed here are generally methylated very frequently in colorectal cancer cell lines and colorectal cancer, while at the same time they are less methylated in normal mucosa from non-cancer donors. It was. These results support the usefulness of the marker gene in the diagnosis of tumors in the respiratory tract-digestive system and in monitoring tumor development.

  Specifically, MAL is 1/23 (4%) of normal mucosal samples from non-cancer donors and 2/21 (10%) of normal mucosal samples taken away from the primary tumor, It is methylated in 45/63 (71%) of adenomas, 49/61 (80%) of carcinomas, and 19/20 (95%) of colon cancer cell lines. Note that the methylation frequency observed for MAL deviates slightly from that seen in Example 1. This deviation is mainly due to the fact that the panel of analyzed samples has been enlarged.

  FBN1 and INA were also rarely methylated in both normal mucosal samples from non-cancer donors and normal mucosal samples taken away from the primary tumor (for FBN1, 1 / 19, 5%, and 2/21, 10%; for INA, 0/21, 0%, and 2/20, 10%, respectively). At the same time, both FBN1 and INA were found in carcinomas (40/49, 82%, and 33/48, 69%, respectively), and adenomas (37/59, 58%, and 31/59, 53%, respectively). It is frequently methylated. The low methylation frequency in both cohorts of normal mucosal samples, and the high methylation frequency in both benign and malignant tumors, suggests that FBN1 and INA are particularly promising for detection of early-onset tumors. At the same time, a test comprising these two markers will most likely have a high specificity.

  SNCA, SPG20, and CNRP1 are generally in carcinomas (37/48 (77%), 44/49 (90%), and 45/48 (94%), respectively) compared to the latter group. As well as higher methylation frequencies in adenomas (42/61 (69%), 48/58 (83%), and 52/59 (88%), respectively). Inclusion of these markers in non-invasive tests can increase sensitivity. In addition to having a low methylation frequency in normal mucosal samples from non-cancer donors, these markers have a relatively high methylation frequency in normal mucosal samples taken away from the primary tumor (respectively 14/21 (67%), (29% -90%), and 9/21 (43%)). This can show a “field effect” around the tumor, where normal appearing cells in the immediate vicinity of the tumor also retain the methylation of these three genes. The presence of such a field effect is non-invasive because more cells that retain SNCA, SPG20, and / or CNRP1 methylation detach into the colon lumen and are excreted with the feces. May enhance the sensitivity of fecal-based tests.

Example 3:
Methylation of marker genes in different samples DNA was purified from stool and MSP was performed on the following genes MAL, FBN1, CNRIP1, INA, SPG20, and SNCA as described in previous examples.

The purification of DNA from 10 stool samples was analyzed for all 6 genes. The methylation status of the corresponding primary tumor was known from 4-5 corresponding tumors.
In addition, blood samples were also collected from 9 out of 10 patients who obtained stool samples, and the results were compared in Table 5.

  DNA was isolated from 250 mg of stool using the QIAamp DNA stool kit (QIAGEN).

  The results of methylation status in genes from different samples are shown in Table 4 below:

  Methylation was detected in all markers in the sample from the stool except for MAL and FBN1. In blood samples, methylation was detected in all genes except FBN1. Overall, sample methylation frequency was high for all genes from blood samples. In particular, the sample methylation frequency in blood samples comprising SNCA and CNRIP1 was very high. Since these markers can be tested by non-invasive procedures such as blood samples, they appear to be particularly well suited as cancer markers.

  DNA was purified from blood (using standard phenol / chloroform method) and MSPs were analyzed for the following genes MAL, CNRIP1, INA, FBN1, SPG20, and SNCA as described in the previous examples. Carried out.

  DNA was purified from 14 blood samples from patients with corresponding primary tumors methylated from all 6 genes.

This example confirms the high sample methylation frequency of genes in blood samples and in particular CNRIP1 and SNCA are very suitable markers for diagnosis and / or screening for cancer or for the development of cancer It is.
SPG20 also appears to be a very promising marker for blood sample screening.

Example 4:
Tissue-specific sample methylation frequency of INA, SNCA, CNRIP1, SPC20, or FBN1 in different cell lines For each sample (cell lines derived from breast, kidney, ovary, pancreas, prostate, uterus and stomach) DNA was bisulfite treated prior to MSP as described in.

  The results of sample methylation frequency of genes in different samples are shown in Table 6 below:

  The promoter methylation status of INA, SNCA, CNRIP1, SPG20, and FBN1 was analyzed using MSP. The methylation frequency was (100%) for all genes tested since all the samples from the gastric cell line were methylated. Overall, INA was methylated in at least one sample of all tested tissues. The highest sample methylation frequency for this gene was found in cell lines from stomach 3/3 (100%), breast 4/6 (66%), and prostate 1/1 (100%). For cell lines from all other tissues, the sample methylation frequency was 50%. SNCA was methylated in cell lines from all examined tissues except the ovary. The highest sample methylation frequency is from stomach 3/3 (100%), breast 6/7 (85%), pancreas 4/6 (66%), and prostate 1/1 (100%) It was a cell line. For the uterus, the sample methylation frequency was 50%. The sample methylation frequency of CNRIP1 was higher in the stomach 3/3 (100%) and in the pancreas (83%) in which 5 out of 6 samples were methylated. SPG20 was methylated in 4 of 6 pancreatic cell lines (66%) and 2 of 4 uterine cell lines (50%). FBN1 was methylated in 4 out of 6 breast cancer cell lines, and 2 out of 4 samples (50%) were methylated in the cell line from the uterus.

  Overall, in the samples from gastric cell lines, all genes were methylated and therefore the sample methylation frequency was high for all genes. In addition, in the breast cell line, all genes were methylated, but the frequency was different among the genes. Furthermore, in the sample from the pancreas, all genes were methylated and for all genes except FBN1 (33%), the frequency was over 50%.

  This experiment clearly suggests that the genes according to the present invention are methylated in cell lines from various cancer tissues and therefore could be used as cancer markers for various cancers. It will be apparent to those skilled in the art that each of the genes according to the present invention can be combined in different ways depending on the type of cancer to be detected. Thus, the gene that showed the best results in the breast cell line would be selected as a marker when detecting breast cancer.

  The results of the tissue specific sample methylation frequency of MAL are listed in Table 8.

Example 5:
Quantitative gene expression analysis was performed for the following genes: SPG20, INA, and CNRIP1.
Gene expression was measured before and after treatment with epigenetic drugs for six colon cancer cell lines. The relative expression levels of SPG20, INA, and CNRIP1 in colorectal cancer cell lines (n = 6) were measured. Expression levels are displayed as fold change calculated from the delta-delta CT method using untreated samples as a reference. The average expression of ACTB and GUSB was used as an endogenous control.

  As previously described, TaqMan real-time fluorescence detection (Applied Biosystems, Foster city, CA) was used to quantify mRNA levels in colon cancer cell lines [Gibson et al. And Heid et al. ]. cDNA was generated from 5 μg of total RNA using a High-Capacity cDNA Archive kit (Applied Biosystems) containing random primers according to the manufacturer's protocol. The genes of interest (SPG20, INA, and CNRIP1), as well as cDNA from endogenous controls (ACTB and GUSB) were separately amplified by 7900HT Sequence Detection System (Applied Biosystems) according to the protocol recommended by the supplier. All samples were analyzed in triplicate. Expression levels were calculated as fold change using the delta-delta CT method using untreated samples as a reference. In order to adjust the possible variable amount of cDNA input in each PCR, we normalized the expression level of the target gene using housekeeping genes ACTB and GUSB.

  For SPG20, INA, and CNRIP1, gene expression is large in the first methylated colon cancer cell lines after demethylation of the promoter caused by the combined treatment with 5-aza-2′-deoxycytidine and trichostatin A. Significantly up-regulated in part (Figures 2, 3, and 4). For INA and CNRIP1, the combination treatment was more effective than the individual treatment with 5-aza-2'-deoxycytidine alone and trichostatin A alone. Combination treatment also enhanced SPG20 expression, however, 5-aza-2'-deoxycytidine treatment alone could achieve similar or higher reactivation. Treatment with the deacetylase inhibitor trichostatin A alone did not enhance either SPG20, INA, or CNRIP1 gene expression. The two doses of 5-aza-2'-deoxycytidine tested here (1 uM and 10 uM) gave similar effects. This means that the demethylation of the cell line is achieved by culturing them in the presence of low doses of 5-aza-2′-deoxycytidine, given the cytotoxicity of this drug, That is an advantage.

  There was a clear correlation between the methylation status and expression of SPG20, INA, and CNRIP1. Thus, for example, methylating the methylation status or level by MSP, and combining the result with the expression level of the corresponding gene may enhance the sensitivity and specificity of the method of the invention.

Example 6:
MAL hypermethylation 65 colorectal cancers from 64 patients (36 with microsatellite stability; MSS and 29 with microsatellite instability; MSI), median size from 52 patients 63 adenomas 8 mm, range 5-50 mm (61 MSS and 2 MSI), 21 normal mucosal samples from 21 colon cancer patients (taken from the distal site of the primary cancer), and 23 normal colorectal mucosa samples from 22 non-cancerous people plus 20 colon cancer cell lines (11 MSS and 9 MSI), and various tissues Patient and cell line DNA from 218 new frozen samples including 29 cancer cell lines from (breast, stomach, kidney, ovary, pancreas, prostate, and uterus; Table 9) were subjected to methylation analysis. The average age at diagnosis was 70 years for patients suffering from carcinoma (range 33-92 years), 67 years for those suffering from adenomas (range 62-72 years), the number of normal mucosa donors One group was 64 years (range 24 to 89 years) and 54 years (range 33 to 86 years) for the second group of normal mucosal donors. Normal samples from colorectal cancer and cancer patients were obtained from a prospective random series collected from seven hospitals located in the southeastern region of Norway. Adenomas were obtained from people who participated in a population-based sigmoidoscopy screening program for colorectal cancer. Normal mucosal samples from persons not afflicted with cancer are obtained from the dead, and the majority (27/44) of the normal population as a whole consisted of mucus only, but the remaining samples from the intestinal wall Collected. Additional clinicopathological data for the current tumor series includes gender and tumor location, as well as polyp size, and total number of polyps per individual for the adenoma series.

  All samples belong to an approved research biobank and are part of a research project approved by national guidelines (Biobank; registered with the Norwegian Institute of Public Health). : Regional Ethics Committee and National Data Inspection Bureau).

  Six colorectal cancer cell lines, HCT15, HT29, SW48, SW480, RKO, and LS1034, were demethylated drugs 5-aza-2′-deoxycytidine (72 μl at 1 μM and 72 h at 10 uM), histone Deacetylase inhibitor trichostatin A (12 h at 0.5 μM) and a combination of both (72 h with 1 μM 5-aza-2′-deoxycytidine, 0.5 μM trichostatin A added to the last 12 h) The processing in was added.

Bisulfite treatment and methylation-specific polymerase chain reaction (MSP)
DNA from primary tumors and normal mucosal samples were treated with bisulfite as previously described. DNA from a colon cancer cell line was treated with bisulfite using the EpiTect bisulfite kit (Qiagen Inc., Valencia, CA, USA). The promoter methylation status of all genes was analyzed by methylation specific polymerase chain reaction (MSP) using HotStarTaq DNA polymerase (Qiagen). All results were confirmed with an independent second round of MSP. Human placental DNA (Sigma Chemical Co, St. Louis, MO, USA) treated with Sss1 methyltransferase (New England Biolabs Inc., Beverly, MA, USA) in vitro was used as a positive control for methylated MSP reaction, In contrast, DNA from normal lymphocytes was used as a positive control for unmethylated alleles. Water was used as a negative control for both reactions. Primers were designed using MethPrimer and Methyl Primer Express and their sequences are listed in Table 7.

Abbreviations: MSP, methylation specific polymerase chain reaction; BS, bisulfite sequencing; M, methylation specific primer; U, unmethylation specific primer; Size, fragment size; An. Temp, annealing temperature (in degrees Celsius). The fragment position is NCBI (RefSeq ID NM 002371), http: // www. ncbi. nlm. nih. gov / mapview / map / search Lists the start and end points (in base-pair units) of each fragment relative to the transcription start provided by cg.

Bisulfite sequencing All colon cancer cell lines (n = 20) were subjected to direct bisulfite sequencing of the MAL promoter. Two fragments were amplified: the A fragment, which spans the points −68 to 168 bases and the fragment B, which spans −427 to −23 bases (overlapping with our MSP product). The A fragment covered a total of 24 CpG sites and was amplified in 35 PCR cycles using HotStarTaq DNA polymerase. Fragment B covered a total of 32 CpG sites and was amplified in 36 PCR cycles using the same polymerase. Primer sequences are listed in Table 8. Excess primers and nucleotides were removed by ExoSAP-IT treatment according to the manufacturer's protocol (GE Healthcare, USE Corporation, Ohio, USA). The purified product was subsequently determined using the AB Prism 3730 sequencer (Applied Biosystems) using the dGTP BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA US A sequence) The approximate amount of methyl cytosine at each CpG site was calculated by comparing the cytosine signal peak height with the sum of the cytosine and thymine signal peak heights as previously described. CpG sites with ratios ranging from 0 to 0.20 are classified as unmethylated, and CpG sites within the range of 0.21 to 0.80 are classified as partially methylated, and 0.81 CpG sites in the range of -1.0 were classified as hypermethylated.

cDNA preparation and real-time quantitative gene expression Total RNA was analyzed using Trizol (Invitrogen, Carlsbad CA, USA) using cell lines (n = 46), tumors (n = 16), and normal tissues (n = 3). And RNA concentrations were measured using ND-1000 Nanodrop (NanoDrop Technologies, Wilmington, DE, USA). For each sample, total RNA was converted to cDNA using a High-Capacity cDNA Archive kit (Applied Biosystems) containing random primers. MAL (Hs00242749 m1 and Hs00360838 m1), endogenous control ACTB (Hs999999993 m1), and GUSB (Hs9999999908) m1) was amplified separately in 96-well first plates according to the recommended protocol (Applied Biosystems) and real-time quantitative gene expression was measured by 7900HT Sequence Detection System (Applied Biosystems). All samples were analyzed in triplicate and the median was used for data analysis. (Contains a mixture of RNAs from 10 different cell lines; Stratagene) Human universal reference RNA was used to generate a standard curve and the resulting quantitative expression levels of MAL Normalized to the mean value of the endogenous control.

Tissue microarray In situ detection of protein expression in colorectal cancer, a tissue microarray (TMA) was constructed based on previously described techniques. Embedded in TMA are 292 cylindrical tissue cores (0.6 mm in diameter) from ethanol-fixed and paraffin-embedded tumor samples from 281 individuals. Samples from the same patient series were examined for various biological variables and clinical endpoints. In addition, the array includes normal tissues from the kidney, liver, spleen, and heart as controls. Separate ethanol-fixed normal colon tissues from 4 individuals with no known history of colorectal cancer were obtained.

Immunohistochemical in situ protein expression analysis 5 μm thick sections of TMA blocks were transferred onto glass slides for immunohistochemical analysis. Sections were deparaffinized in a xylene bath for 10 minutes and rehydrated with a series of graded ethanol baths. Heat-induced epitope repair was heated at maximum effect (850 W) by microwave for 15 minutes while immersed in 10 mM citrate buffer pH 6.0 containing 0.05% Tween-20, followed by 100 W This was carried out by heating for 5 minutes. After cooling to room temperature, immunohistochemical staining was performed according to the protocol of the DAKO Envision + ™ K5007 kit.

  The primary antibody, mouse clone 6D9 anti-MAL, was used at a dilution of 1: 5000, which allowed staining of kidney tubules as a positive control while leaving myocardial tissue unstained as a negative control. Slides were counterstained with hematoxylin for 2 minutes and then dehydrated with increasing degrees of ethanol and finally xylene. Results from immunohistochemistry were obtained by independent scoring by one of the authors and reference pathologists.

Statistics All P values were obtained from two-tailed statistical tests using SPSS 13.0 software (SPSS, Chicago, IL, USA). Fisher's exact test was used to analyze the 2 × 2 contingency table. A 2 × 3 table and chi-square test were used to analyze the potential association between MAL quantitative gene expression and promoter methylation status. Samples were divided into two categories according to their gene expression level: low expression includes samples with comparable or low gene expression compared to the median across all cell lines or all tumors, and high expression. Included samples with higher gene expression compared to the median. The methylation situation was divided into three categories: unmethylated, partially methylated, and hypermethylated.

MAL promoter methylation status in tissues and cell lines The MAL promoter methylation status was analyzed using MSP (FIG. 5). One of the normal mucosal samples from 4 non-cancer donors (4%) and 2 of the normal mucosal samples taken away from the 21 primary tumors (10%) were methyl However, only a low intensity band was shown after gel electrophoresis compared to the positive control. Of the 63 adenomas, 45 (71%) and 49/61 carcinomas (80%) showed promoter hypermethylation. 19 of 20 colon cancer cell lines (95%) and 15/26 (58%) of cancer cell lines from various tissues (breast, kidney, ovary, pancreas, prostate and uterus) Were hypermethylated (Table 9 lists tissue-specific frequencies).

  The promoter methylation status of individual cell lines was assessed by methylation specific polymerase chain reaction (MSP). The methylation frequency reflects the number of methylated (M and U / M) samples from each tissue. Abbreviations: U, unmethylated; M, methylated.

  The hypermethylation frequency seen in normal samples was significantly lower than that in adenomas (P <0.0001) and carcinomas (P <0.0001). Hypermethylation of the MAL promoter was not related to MSI status, gender, or age, nor was it associated with malignant or benign tumors. In common with carcinomas, tumors distal to the intestinal tract (left and rectum) were frequently hypermethylated compared to tumors proximal but were not statistically significant (P = 0.0). 088). In common with adenomas, no significant relationship could be found between MAL promoter methylation status and polyp size or number.

Verification of MAL promoter methylation status by bisulfite sequencing Two overlapping fragments of the MAL promoter were bisulfite sequenced in 20 colon cancer cell lines. The results are summarized in FIG. 6, and representative raw data can be seen in FIG. And a high association was seen between the methylation status assessed by MSP and bisulfite sequencing of overlapping fragment A. However, fragment B had only a low association with MSP data. For this fragment located more upstream relative to the transcription start point, several consecutive CpG sites were frequently unmethylated and / or partially methylated. This was also true in cell lines that were shown to be heavily methylated around the transcription start point (fragment A; FIG. 6).

Real-time quantitative gene expression The level of MAL mRNA expression in cell lines (n = 46), primary colorectal cancer (n = 16), and normal mucosa (n = 3) was assessed by quantitative real-time PCR. In common cell lines, there was a strong association between hypermethylation of the MAL promoter and decreased or lost gene expression (P = 0.041; FIG. 8). Furthermore, MAL gene expression was up-regulated in colon cancer cell lines after demethylation of the promoter induced by the combined treatment of 5-aza-2′deoxycytidine and trichostatin A (FIG. 9). Treatment with the deacetylase inhibitor trichostatin A alone did not enhance MAL expression, whereas treatment with 5-aza-2'deoxycytidine, which demethylates DNA, caused high expression in HT29 cells. However, it caused a more gradual level in HCT15 cells (FIG. 9). In common with primary colorectal cancer, those that retain MAL promoter hypermethylation (n = 13) express only slightly lower levels of MAL mRNA compared to unmethylated tumors (n = 3). Was not statistically significant (Figure 8).

MAL protein expression is lost in colorectal cancer.
To evaluate the immunohistochemical analysis of MAL, kidney and myocardial tissues were included as positive and negative controls, respectively (FIGS. 10A-B). From 231 scorable colorectal tissue cores, ie, those containing malignant colorectal epithelial tissue, 198 were negative for MAL staining (FIGS. 10C-D). Of these, 29 had positive staining for non-epithelial tissue components within the same tissue core, mainly neurons and blood vessels (not shown). In contrast, all sections of normal colon tissue contained positive MAL staining in epithelial cells (FIGS. 10E-F).

  These experiments indicate that the MAL promoter near the transcription start point is hypermethylated in most malignant as well as benign colorectal tumors, in contrast to normal unmethylated intestinal mucosal cell samples. And we still argue that MAL is a promising diagnostic biomarker for early colorectal tumorigenesis. In addition, hypermethylated MAL was found in cancer cell lines from breast, kidney, ovary, and uterus.

  Others have previously shown that hypermethylation of MAL by quantitative methylation-specific polymerase chain reaction (MSP) is present only in a small portion (6%, 2/34) of colon carcinomas. (Mori et al.). In contrast, Applicants have now demonstrated a significantly higher methylation frequency of MAL (71% in adenomas and 80% in carcinomas) in both benign and malignant colorectal tumors. The discrepancy in methylation frequency between this report and previous studies by Mori et al. Is probably the result of the study design. From direct bisulfite sequencing of colorectal cancer cell lines we showed here that MAL DNA methylation is heterogeneously distributed within the CpG island of its promoter (FIG. 6). CpG islands are often more extensive than a kilobase of gene promoters, and methylation status within this region is sometimes mistakenly recognized as uniformly distributed. Since the results of MSP analysis depend on unmethylated and methylated primer sequence matches or mismatches to the bisulfite treated DNA, one skilled in the art will ensure that the primers anneal to the relevant CpG sites within the gene promoter. Should. In this study, Applicants designed an MSP primer near the transcription start of the gene (−72 to +70) and the overall methylation status of MAL as assessed by MSP and our MSP primer Agreement between the methylation status of individual CpG sites covered by the set was obtained by bisulfite sequencing (Figure 6). This part of the CpG island was hypermethylated in the majority (95%) of colon cancer cell lines. We also show that these cell lines, as well as those from other tissues, show a loss of MAL RNA expression from quantitative real-time analysis and DNA by combined treatment with 5-aza-2'deoxycytidine and trichostatin A It was found that removal of hypermethylation reinduces the expression of MAL in colon cancer cell lines (FIG. 9). Furthermore, by analyzing a large series of clinically representative samples by protein immunohistochemistry, we found that MAL expression is lost in malignant colorectal epithelial cells compared to normal mucosa. It was confirmed.

  The inventor further analyzed the same MAL promoter region as Mori et al., Located at −206 to −126 base pairs upstream of the transcription start point. By direct bisulfite sequencing, we found 19 colon cancer cells in which only a small number of CpG sites targeted by the Mori antisense primer were heavily methylated around the transcription start point. It was shown to be methylated in the strain (FIG. 6). Therefore, we found that the very low (6 percent) methylation frequency (Mori et al.) First reported for MAL in colon carcinomas is most likely due to the influence of primer design and selection of CpG sites to be investigated. The conclusion was high.

  Inactivation of hypermethylation of the MAL promoter may also be common for other cancer types. In this study, hypermethylated MAL was found in cancer cell lines from breast, kidney, ovary, and uterus.

  This analysis on cancer cell lines from seven tissues suggests that hypermethylation in a limited region near the transcription start of MAL is associated with decreased or lost gene expression.

  A highly sensitive non-invasive screening approach for colorectal cancer will significantly improve clinical outcomes for patients. Such a diagnostic test will in principle measure the status of a single biomarker.

  Hypermethylation of the MAL promoter represents a frequently hypermethylated gene in precancerous colorectal lesions, which coincides with a low methylation frequency in normal intestinal mucosal cells. The presence of such epigenetic changes in pre-cancerous tissue may also have implications for cancer chemoprevention. Suppressing or reversing these epigenetic changes may prevent progression to a malignant phenotype (Kopelovich et al.). MAL promoter hypermethylation remains one of the most promising diagnostic biomarkers for early detection of colorectal tumors.

References

Claims (11)

  1. A method of indicating whether a subject has developed, is or is likely to develop cancer, or has recurred after cancer treatment of the respiratory tract-digestive system, the following steps:
    a) measuring the methylation level of the CpG site, the number of methylated CpG sites, or the methylation status within the nucleic acid sequence of the SPG20 promoter region, first exon or intron in the sample obtained from the above-mentioned subject;
    b) comparing said methylation level, number of methylated CpG sites, or methylation status of CpG sites with reference; and c) said methylation level, number of methylated CpG sites, or CpG If the methylation status of the site is high compared to the above criteria, the subject is likely to develop, is or is likely to develop cancer, or has recurred after cancer treatment And if the methylation level, the number of methylated CpG sites, or the methylation status of the CpG sites is below the above criteria, the subject is less likely to develop cancer or develop Said method comprising: indicating that it has not, is unlikely to develop, or has not recurred after cancer treatment.
  2. A method of indicating whether a subject has developed, is or is likely to develop cancer, or has recurred after cancer treatment of the respiratory tract-digestive system, the following steps:
    a) measuring the SNCA promoter region, the methylation level of the CpG site in the nucleic acid sequence of the first exon or intron, the number of methylated CpG sites, or the methylation status in a sample obtained from the above-mentioned subject;
    b) comparing said methylation level, number of methylated CpG sites, or methylation status of CpG sites with reference; and c) said methylation level, number of methylated CpG sites, or CpG If the methylation status of the site is high compared to the above criteria, the subject is likely to develop, is or is likely to develop cancer, or has recurred after cancer treatment And if the methylation level, the number of methylated CpG sites, or the methylation status of the CpG sites is below the above criteria, the subject is less likely to develop cancer or develop Said method comprising: indicating that it has not, is unlikely to develop, or has not recurred after cancer treatment.
  3. A method of indicating whether a subject has developed, is or is likely to develop cancer, or has recurred after cancer treatment of the respiratory tract-digestive system, the following steps:
    a) measuring the methylation level of the INA promoter region, the first exon or intron nucleic acid sequence, the number of methylated CpG sites, or the methylation status in the sample obtained from the above-mentioned subject,
    b) comparing said methylation level, number of methylated CpG sites, or methylation status of CpG sites with reference; and c) said methylation level, number of methylated CpG sites, or CpG If the methylation status of the site is high compared to the above criteria, the subject is likely to develop, is or is likely to develop cancer, or has recurred after cancer treatment And if the methylation level, the number of methylated CpG sites, or the methylation status of the CpG sites is below the above criteria, the subject is less likely to develop cancer or develop Said method comprising: indicating that it has not, is unlikely to develop, or has not recurred after cancer treatment.
  4. The methylation level, the number of methylated CpG sites, or the methylation status of CpG sites can be determined by bisulfite sequencing, quantitative and / or qualitative methylation specific polymerase chain reaction (MSP). , Pyrosequencing, Southern blotting, restriction enzyme landmark genome scanning (RLGS), single nucleotide primer extension, CpG island microarray, SNUPE, COBRA, mass spectrometry, methylation-specific restriction enzymes The method according to any one of claims 1 to 3 , which is measured by use, measurement of the expression level of the gene, or a combination thereof.
  5. The methylation specific PCR is two CpG sites, and comprising the use of nucleic acid primers capable of hybridizing to a nucleic acid sequence comprising no cytosine residues in CpG sites, according to claim 1-4 The method according to any one of the above.
  6. The cancer is: colorectal tumor, lung tumor, small cell lung cancer, non-small cell lung cancer, esophageal tumor, stomach tumor, pancreatic tumor, liver tumor, gallbladder and / or bile duct tumor, small intestine The method according to any one of claims 1 to 5 , wherein the method is selected from the group consisting of:
  7. The sample is obtained from blood, stool, urine, pleural effusion, bile, bronchial fluid, gargle, tissue biopsy, ascites, pus, cerebrospinal fluid, puncture fluid, follicular fluid, tissue, or mucus. 7. The method according to any one of items 6 .
  8. Methylation level, the number of methylated CpG sites or methylation status of CpG sites is combined with at least one additional marker method according to any one of claims 1-7.
  9. A method of indicating whether a subject has developed, is or is likely to develop cancer, or has recurred after cancer treatment of the respiratory tract-digestive system, the following steps:
    a) In a sample obtained from the above-mentioned subject:
    1) a nucleic acid sequence defined by any of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 16;
    2) a nucleic acid sequence that is complementary to the sequence defined in 1);
    3) A partial sequence of the nucleic acid sequence defined in 1) or 2);
    4) Methylation level within a nucleic acid sequence comprising a sequence selected from the group consisting of nucleic acid sequences that are at least 90% identical to the sequence defined in 1), 2), or 3), methylated CpG Measuring the number of sites or methylation status of CpG sites,
    b) comparing said methylation level, number of methylated CpG sites, or methylation status of CpG sites with reference; and c) said methylation level, number of methylated CpG sites, or CpG If the methylation status of the site is high compared to the above criteria, the subject is likely to develop, is or is likely to develop cancer, or has recurred after cancer treatment And if the methylation level, the number of methylated CpG sites, or the methylation status of the CpG sites is below the above criteria, the subject is less likely to develop cancer or develop Said method comprising: indicating that it has not, is unlikely to develop, or has not recurred after cancer treatment.
  10. 10. The method according to claim 9 , wherein the nucleic acid sequence in step 4) is at least 95% identical to the sequence defined in 1), 2) or 3).
  11. Methylation as an indicator of whether a subject has, is developing or is likely to develop airway-digestive cancer, or whether the subject has relapsed after airway-digestive cancer treatment Use of any of SPG20, INA, and SNCA in a diagnostic assay to assess level, number of methylated CpG sites, or methylation status of CpG sites.
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